Svante Norgren

The mutation spectrum of the facilitative glucose transporter gene SLC2A2 (GLUT2) in patients with Fanconi-Bickel syndrome

Abstract. We report a total of 23 novel mutations of the SLC2A2 (GLUT2) gene in 49 patients with a clinical diagnosis of Fanconi-Bickel syndrome (FBS). Molecular genetic analysis has now been performed in more than 50% of the 109 FBS cases from 88 families that we have been able to locate world-wide since the original report in 1949. In these 49 patients, 33 different SLC2A2 mutations (9 missense, 7 nonsense, 10 frameshift, 7 splice-site) have been detected. Thus, our results confirm that mutations of SLC2A2 are the basic defect in patients with FBS. Mutations of SLC2A2 were detected in historical FBS patients in whom some of the characteristic clinical features (hepatorenal glycogen accumulation, glucose and galactose intolerance, fasting hypoglycemia, a characteristic tubular nephropathy) and the effect of therapy were described for the first time. Mutations were also found in patients with atypical clinical signs such as intestinal malabsorption, failure to thrive, the absence of hepatomegaly, or renal hyperfiltration. No single prevalent SLC2A2 mutation was responsible for a significant number of cases. In a high percentage (74%) of FBS patients, the mutation is homozygous, so we conclude that the prevalence of SLC2A2 mutations is relatively low in most populations. No mutational hot spots within SLC2A2 or even within homologous sequences among the genes for facilitative glucose transporters were detected.

Physiological and pathological aspects of GSH metabolism

The antioxidant glutathione is found in low levels in diseases in which increasing evidence implicate oxidative stress in the development of the disease, for example retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, patent ductus arteriosus and asthma. Glutathione is metabolized in the γ‐glutamyl cycle, which involves six different enzymes. The synthesis of glutathione is a two‐step process in which the first step is catalysed by γ‐glutamylcysteine synthetase and the second step by glutathione synthetase. Glutathione synthetase deficiency is an autosomal recessive disease and the most common inborn error of the γ‐glutamyl cycle. Approximately 25% of patients with hereditary glutathione synthetase deficiency die during childhood. Patients present with a clinical picture ranging from compensated haemolytic anaemia to a complex disorder with additional symptoms like 5‐oxoprolinuria, metabolic acidosis and central nervous system impairment. Even though the correlation between phenotype and genotype in these patients is complex, an indication of the phenotype can be based on the type of mutation involved. Also, there is a correlation between the glutathione synthetase activity and the level of glutathione in cultured fibroblasts. Inborn errors have also been described in three additional steps of the γ‐glutamyl cycle, namely γ‐glutamyltranspeptidase, 5‐oxoprolinase and γ‐glutamylcysteine synthetase.

Orlistat treatment in obese prepubertal children: a pilot study

Aim: This study investigated orlistat treatment in obese prepubertal children with regard to tolerance, safety and psychological well‐being. Methods: 11 healthy, severely obese prepubertal children (age 8.3–12.3 y, body mass index standard deviation score 5.3–9.2) were recruited for a 12wk open treatment. Before, during and after treatment, the participants were investigated by psychological evaluation, blood chemistry, and parameters reflecting obesity and fat mass. Results: The participants were able to comply with the treatment, as indicated by pill counts and self reports, and expressed a desire to continue the treatment after the study period. Gastrointestinal side effects were mild and tolerable. No negative effects on psychological or physical well‐being were detected, and the psychological evaluation demonstrated increased avoidance of fattening food, body shape preoccupation and oral control (p= 0.011). The median weight loss was 4.0 kg (range –12.7 to +2.5 kg, p= 0.016) and was highly correlated to decreased fat mass (regression coefficient 0.953, p < 0.01).

Dental aberrations in children and adolescents with osteogenesis imperfecta

The aim of this investigation was to study dental aberrations in a large sample of unrelated patients with different types and forms of osteogenesis imperfecta (OI). Sixty-eight non-related index patients aged 0.3 to 20 years (mean, 10 years) were examined clinically. Panoramic radiographs from 49 patients were analyzed. Dentinogenesis imperfecta (DI) type I was found in 27 of 65 patients and was significantly more common in OI type III than in types I and IV and in patients with a severe form of the disease. The presence or absence of DI showed almost complete accordance between affected parents and children and between affected siblings. Moreover, agenesis was found in 11 of 49 patients, apically extended pulp chambers in 20 of 48 patients, and impaction of second permanent molars in 7 of 19 patients older than 15 years. The percentage of patients with no apparent dental aberrations was approximately the same in patients with OI type I and type III and in patients with mild and severe form of the disease. The high prevalence of dental aberrations in OI stresses the importance of clinical and radiographic odontologic examination as part of the clinical investigation. In patients with mild forms of the disease, in whom the medical diagnosis is uncertain, demonstration of disturbances in dental development can be crucial for establishing the OI diagnosis.

Differences in the Ratio of RNA Encoding Two Isoforms of the Insulin Receptor Between Control and NIDDM Patients: The RNA Variant Without Exon 11 Predominates in Both Groups

Two alternative forms of the insulin receptor with different affinities for insulin are expressed as a result of alternative splicing of RNA corresponding to exon 11 of the IR gene. The percentage of IR-RNA molecules without exon 11, encoding the high-affinity isoform, was determined by cDNA-mediated PCR amplification of RNA extracts from the quadriceps femoris muscle of healthy control subjects (n = 9) and NIDDM patients (n = 7). In both patients and control individuals, a majority of the IR-RNA molecules contained exon 11. In addition, the proportion of IR-RNA molecules without exon 11 was decreased in patients (21 ± 1%) compared with control subjects (31 ± 3%) (P = 0.018). Careful investigation of the kinetics of the PCR-based assay system, as well as the conditions for separation of the PCR products, allowed us to suggest a possible explanation of the discrepant results concerning the alternative splicing presented in previous reports. The diabetic subjects as a group had higher fasting insulin levels and lower insulin-mediated glucose uptake during a euglycemic-hyperinsulinemic clamp (P = 0.042). However, identification of the regulatory pathways leading to the splicing alteration in NIDDM patients requires further investigation.

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and γ-glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or γ-glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.

N-Acetylcysteine Amide Protects Renal Proximal Tubular Epithelial Cells against Iohexol-Induced Apoptosis by Blocking p38 MAPK and iNOS Signaling

Background: The pathogenesis of contrast-induced nephropathy (CIN) is still poorly understood and apoptosis via oxidative stress has been proposed as one possible mechanism. We therefore studied the apoptotic signaling mechanism in CIN and also tested whether the new antioxidant N-acetylcysteine amide (NACA) could prevent CIN. Methods: LLC-PK1 cells were exposed to a widely used contrast agent, iohexol (IH). Cytotoxicity was assessed with morphology and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell death was analyzed by the DNA content analysis and PARP cleavage. Protein expression was assessed with Western blotting. Results: We observed cell death with apoptotic features in a dose- and time-dependent manner. Initiation of IH-induced apoptosis was mediated by upregulation of Bax and downregulation of Bcl-2 and Mcl-1, which was preceded by p38 MAPK activation and iNOS induction. Inhibitors of p38 MAPK and iNOS partially abolished IH-induced apoptosis. Furthermore, we found pretreatment with NACA partially protected cells from IH-induced death by reverting the expression of Bcl-2, Mc1-1 and Bax expression through inhibition of p38 MAPK and iNOS pathway. Conclusions: This study demonstrates that apoptosis occurs during CIN. Apoptosis is associated with activations of p38 MAPK and iNOS. Pretreatment with the antioxidant NACA could prevent IH-induced cell death by blocking the p38 MAPK/iNOS signaling pathway.

Growth Hormone Treatment Downregulates Serum Leptin Levels in Children Independent of Changes in Body Mass Index

The changes in serum leptin levels during growth hormone (GH) treatment were studied in 27 children, 17 with GH deficiency (GHD), 10 with idiopathic short stature (ISS), and 9 with Prader-Willi syndrome (PWS). Within 1 month of GH treatment, serum leptin levels decreased by 40% in the GHD children (p < 0.01). There was no significant change in serum leptin level in the children with ISS. In children with PWS, the mean serum leptin level decreased by almost 60% after 3 months of treatment (p < 0.001). Thereafter, no further decline was observed in any of the 3 groups. Changes in body composition became evident first after the 3 months of treatment. In the GHD children, the BMI was unchanged while the mean body fat percentage was 2.7% lower after 1 year of GH treatment (p < 0.05). In the ISS children, neither BMI nor body fat percentage were significantly changed during treatment. The PWS children exhibited a significant decrease in BMI after 6 months of GH treatment without any further change during the remaining period of treatment. In this group, the mean body fat percentage decreased from 42 ± 2.4 to 28 ± 2.2% after treatment (p < 0.001). The finding that the fall in leptin occurs before changes in body composition become detectable suggests a direct effect of GH on leptin production, metabolism, or clearance.

Glutathione synthetase deficiency: Is γ-glutamylcysteine accumulation a way to cope with oxidative stress in cells with insufficient levels of glutathione?

Glutathione (GSH) plays a major role in the cellular defence against oxidative stress and other vital cellular functions. It therefore seems inevitable that patients with severe depletion of GSH will not survive. However, at least some with glutathione synthetase (GS) deficiency do. This study was done to determine whether these patients have a mechanism to compensate for their GSH deficiency. Cell-free extracts of cultured fibroblasts from 9 patients with GS deficiency and 9 control subjects were analysed by HPLC for low-molecular-weight thiol compounds. The patients′ cells contained 7.4 nmol of GSH per mg of protein (median; range 2.8–25.2) compared to 33.0 nmol in control fibroblasts (range 26.7–51.4) (p<0.01). On the other hand, the patients′ cells accumulated 18.1 nmol of γ-glutamylcysteine (γ-GC) per mg of protein (median; range 6.9–71.7), whereas the control cells contained 0.1 nmol (range 0.05–0.16) (p<0.01). The cysteine concentrations in the patients′ cells were 20.7 nmol/mg protein (median; range 9.4–52.9) compared to 8.9 nmol in control cells (range 3.0–12.4) (p<0.01). Cultured fibroblasts from patients with GS deficiency have low levels of GSH, but instead accumulate γ-GC. We suggest that γ-GC, which contains both reactive groups of GSH (i.e. the sulphydryl and γ-glutamyl groups), can compensate for GSH in the cellular defence against oxidative stress. Thus, γ-GC may alleviate, but only partly prevent, serious consequences of insufficient GSH levels in affected patients. Since the sum of the levels of GSH and γ-GC in GS-deficient cells (median 31.5 nmol/mg protein, range 16.2–79.0) was similar to the level of GSH alone incontrol cells (33.0 nmol/mg protein, range 26.7–51.4), we propose that the cultured fibroblasts may have a mechanism to regulate in a coordinated way the levels of GSH and γ-GC; for instance, by both compounds acting as feedback inhibitors of γ-GC synthetase.

Glucose metabolism and body composition in young adults treated with TBI during childhood.

After SCT in childhood, survivors may develop disorders of glucose metabolism. The role of obesity is controversial. We measured insulin sensitivity using the homeostasis model assessment (HOMA) and the frequently sampled i.v. glucose tolerance test (FSIVGTT), as well as body composition using dual-energy X-ray absorptiometry in 18 young adults median 18.2 years after SCT and compared them with matched controls. We also measured growth hormone (GH) secretion, and levels of leptin and adiponectin. HOMA showed insulin resistance in eight patients (44%), as opposed to none of the controls (P=0.008) and FSIVGTT showed a decreased sensitivity index in the patients (2.98 vs 4.54 mU/L/min, P=0.042). Dual energy X-ray absorptiometry showed a higher percentage fat mass in the patients (34.9 vs 24.3%, P=0.011), which correlated inversely with the sensitivity index (r=−0.52, P=0.032). The patients had a lower peak value of GH (GHmax 9 vs 20.7 mU/L, P=0.002). Time post SCT correlated with percentage fat mass and inversely with GHmax. The patients had higher levels of leptin and lower levels of adiponectin, even after adjustment for fat mass. We propose that the decreased insulin sensitivity may primarily be explained by the adverse body composition, which may owe to long-standing GH deficiency.