Inhibitory effect of pax4 on the human insulin and islet amyloid polypeptide (IAPP) promoters.

Pax4 is a paired-box transcription factor that plays an important role in the development of pancreatic beta-cells. Two Pax4 cDNAs were isolated from a rat insulinoma library. One contained the full-length sequence of Pax4. The other, termed Pax4c, was identical to Pax4 but lacked the sequences encoding 117 amino acids at the COOH-terminus. Pax4 was found to inhibit the human insulin promoter through a sequence element, the C2 box, located at -253 to -244, and the islet amyloid polypeptide promoter through a sequence element located downstream of -138. The inhibitory activity of Pax4 was mapped to separate regions of the protein between amino acids 2-230 and 231-349.

Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells.

One of the mechanisms whereby glucose stimulates insulin gene transcription in pancreatic beta-cells involves activation of the homeodomain transcription factor PDX1 (pancreatic/duodenal homeobox-1) via a stress-activated pathway involving stress-activated protein kinase 2 (SAPK2, also termed RK/p38, CSBP, and Mxi2). In the present study we show, by Western blotting and electrophoretic mobility shift assay, that in human islets of Langerhans incubated in low glucose (3 mM) PDX1 exists as an inactive 31-kDa protein localized exclusively in the cytoplasm. Transfer of the islets to high (16 mM) glucose results in rapid (within 10 min) conversion of PDX1 to an active 46-kDa form that was present predominantly in the nucleus. Activation of PDX1 appears to involve phosphorylation, as shown by incorporation of 32Pi into the 46-kDa form of the protein. These effects of glucose could be mimicked by chemical stress (sodium arsenite), or by overexpression of SAPK2 in the beta-cell line MIN6. Overexpression of SAPK2 also stimulated PDX1-dependent transcription of a -50 to -250 region of the human insulin gene promoter linked to a firefly luciferase reporter gene. The effects of glucose were inhibited by the SAPK2 inhibitor SB 203580, and by wortmannin and LY 294002, which inhibit phosphatidylinositol 3-kinase, although the effects of stress (arsenite) were inhibited only by SB 203580. These results demonstrate that glucose regulates the insulin gene promoter through activation and nuclear translocation of PDX1 via the SAPK2 pathway.

Impaired expression of transcription factor IUF1 in a pancreatic beta-cell line derived from a patient with persistent hyperinsulinaemic hypoglycaemia of infancy (nesidioblastosis).

Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI), or nesidioblastosis, is a rare disorder which may be familial or sporadic, and which is characterized by unregulated secretion of insulin and profound hypoglycaemia in the neonate. The defect has been linked in some patients to mutations in the sulphonyl urea receptor gene (SUR). The present study investigated potential defects in the regulation of the insulin gene by glucose in a beta-cell line (NES 2Y) derived from a patient with PHHI. The results show that the insulin promoter is unresponsive to glucose in PHHI, and that this defect can be attributed to impaired expression of the transcription factor IUF1. Because IUF1 is involved not only in linking glucose metabolism to the control of the insulin, but is also a major regulator of beta-cell differentiation during embryogenesis, we propose that impaired expression of IUF1 contributes to beta-cell dysfunction in PHHI by leading to abnormal beta-cell differentiation.

Fucosidosis: genetic and biochemical analysis of eight cases.

The molecular basis of the deficiency of alpha-L-fucosidase has been investigated in eight patients who had been diagnosed clinically and enzymatically as suffering from the autosomal recessive lysosomal storage disease fucosidosis. None of the patients had a deletion or gross alteration of the alpha-L-fucosidase gene (FUCA1). Single strand conformation polymorphism (SSCP) analysis followed by direct sequencing of amplified exons and flanking regions identified putative disease causing mutations in six of the patients, who had severe forms of the disease and very low residual alpha-L-fucosidase activity and protein. They were a 10 bp deletion in exon 1 (E113fs), a 1 bp deletion at position -2 of intron 2 (S216fs), a g-->a transition at IVS5+1, point mutations W183X and N329Y in exons 3 and 6, respectively, and a compound allele consisting of a point mutation in the signal peptide in exon 1, P5R, and a 1 bp insertion in exon 6 (Y330fs). One patient in whom an SSCP change was not detected had residual alpha-L-fucosidase activity and cross reacting protein in the heterozygous range and normal metabolism of metabolites containing fucose in his fibroblasts, consistent with the low activity polymorphism. The eighth patient, who had a partial deficiency of alpha-L-fucosidase in her fibroblasts and leucocytes at a young age but normal alpha-L-fucosidase activity and protein at a later age, was homozygous for the common Q281R polymorphism in exon 5. She had no other sequence changes and Kivlin (Peters plus) syndrome has subsequently been diagnosed. The basis of her transient deficiency of alpha-L-fucosidase is not known. The detection of five novel mutations in six severely affected patients confirms the genetic heterogeneity in fucosidosis.

Constitutional nonsense germline mutations in the RB1 gene detected in patients with early onset unilateral retinoblastoma.

The 'two-hit' hypothesis for the development of the childhood eye cancer, retinoblastoma (Rb), predicts that bilaterally affected individuals will carry germline mutations. The second suggestion is that patients with early presentation of unilateral tumours also carry predisposing mutations. We have used SSCP analysis to study the 27 individual exons of the RB1 gene in constitutional DNA from 3 patients whose tumours were treated under the age of 12 months. Bandshifts on SSCP gels were detected in 2 of these patients which, on sequencing, were shown to be a C-->T transition converting a CGAarg to a TGAstop codon in exon 17 and an 8 bp deletion in exon 20 resulting in a downstream stop codon. The mutations seen in these patients are reminiscent of those seen in patients with hereditary Rb and confirms that at least some early onset unilateral cases carry constitutional mutations, which has important implications for genetic screening and counselling of these individuals.

Allogeneic bone marrow transplantation for fucosidosis.

Bone marrow transplantation was performed on an 8-month-old boy who was diagnosed as having fucosidosis following the diagnosis of the disease in his older brother. Although he was asymptomatic and his development was normal, abnomalities were found on an MRI scan prior to transplant. In the absence of a suitable related donor, an unrelated volunteer donor was used. Conditioning for the transplant consisted of busulphan and cyclophosphamide. Graft-versus-host disease prophylaxis consisted of in vitro T cell-depletion of the bone marrow and in vivo administration of cyclosporin. The post-transplant period was complicated by moderately severe graft-versus-host disease. Engraftment was documented by the presence of donor levels of alpha-fucosidase, donor blood group and tissue type (difference in the DQ antigen), and chromosomal polymorphism pattern of donor origin. Eighteen months after transplant, there is evidence of mild neurodevelopmental delay. By contrast, his elder sibling showed far greater developmental delay at the same age. The patient's MRI scan shows improvement. We believe this to be the first case of human fucosidosis treated by bone marrow transplantation.

A 5' splice site mutation in fucosidosis.

Fucosidosis is a rare, autosomal recessive, lysosomal storage disease, resulting from a deficiency of the enzyme alpha-fucosidase (EC 3.2.1.51). It is characterised clinically by progressive mental and motor deterioration, growth retardation, coarse facies, and often recurrent infections, but the course of the disease is variable. The gene encoding lysosomal alpha-fucosidase has been mapped to the short arm of chromosome 1 at position 1p34.1-36.1 and has been called FUCA1. Two mutations causing disease have been described previously, a C-->T change in exon 8 giving rise to a premature, in frame TAA stop codon, and a deletion of at least two exons from the 3' end of the gene. In this paper we present evidence that a homozygous G-->A transition in the first position of the 5' splice site of intron 5 of FUCA1 is the disease causing mutation in a 9 year old child of distantly related parents. A new banding pattern was detected in the patient by Southern blotting of genomic DNA using TaqI restriction and a cDNA FUCA1 probe. The patient was homozygous for this pattern. Three sibs with alpha-fucosidase activity below the normal reference range and both parents were heterozygous. This pattern was not detected in 26 other fucosidosis patients and has not been found in any controls. The mutation was localised by a combination of restriction mapping using different cDNA probes, single stranded conformational polymorphism analysis of exons and flanking regions amplified by the polymerase chain reaction, and by direct sequencing of the amplified sequence. A view of the nature of the mutation, its cosegregation with the disease mutation and its absence in controls, it is probable that the 5' splice site mutation causes fucosidosis in this child.