Reduced display of conformational epitopes in the N-terminal truncated GAD65 isoform: relevance for people with stiff person syndrome or DQ8/8-positive Type 1 diabetes mellitus.

AIMS: To investigate whether the N-terminal truncated glutamic acid decarboxylase 65 (GAD65) isoform is as well recognized by people with stiff person syndrome as it is by people with Type 1 diabetes, and whether conformational GAD65 antibody epitopes are displayed properly by the isoform. METHODS: GAD65 antibody-positive healthy individuals (n=13), people with stiff-person syndrome (n=15) and children with new-onset Type 1 diabetes (n=654) were analysed to determine binding to full-length GAD65 and the N-terminal truncated GAD65 isoform in each of these settings. GAD65 autoantibody epitope specificity was correlated with binding ratios of full-length GAD65/N-terminal truncated GAD65. RESULTS: The N-terminal truncated GAD65 isoform was significantly less recognized in GAD65Ab-positive people with stiff-person syndrome (P=0.002) and in healthy individuals (P=0.0001) than in people with Type 1 diabetes. Moreover, at least two specific conformational GAD65Ab epitopes were not, or were only partially, presented by the N-terminal truncated GAD65 isoform compared to full-length GAD65. Finally, an N-terminal conformational GAD65Ab epitope was significantly less recognized in DQ8/8 positive individuals with Type 1 diabetes (P=0.02). CONCLUSIONS: In people with stiff person syndrome preferred binding to the full-length GAD65 isoform over the N-terminal truncated molecule was observed. This binding characteristic is probably attributable to reduced presentation of two conformational epitopes by the N-terminal truncated molecule. These findings support the notion of disease-specific GAD65Ab epitope specificities and emphasize the need to evaluate the applicability of novel assays for different medical conditions.

Doubly Reactive INS-IGF2 Autoantibodies in Children with Newly Diagnosed Autoimmune (type 1) Diabetes.

The splice variant INS-IGF2 entails the preproinsulin signal peptide, the insulin B-chain, eight amino acids of the C-peptide and 138 unique amino acids from an ORF in the IGF2 gene. The aim of this study was to determine whether levels of specific INS-IGF2 autoantibodies (INS-IGF2A) were related to age at diagnosis, islet autoantibodies, HLA-DQ or both, in patients and controls with newly diagnosed type 1 diabetes. Patients (n = 676), 0-18 years of age, diagnosed with type 1 diabetes in 1996-2005 and controls (n = 363) were analysed for specific INS-IGF2A after displacement with both cold insulin and INS-IGF2 to correct for non-specific binding and identify double reactive sera. GADA, IA-2A, IAA, ICA, ZnT8RA, ZnT8WA, ZnT8QA and HLA-DQ genotypes were also determined. The median level of specific INS-IGF2A was higher in patients than in controls (P < 0.001). Irrespective of age at diagnosis, 19% (126/676) of the patients had INS-IGF2A when the cut-off was the 95th percentile of the controls (P < 0.001). The risk of INS-IGF2A was increased among HLA-DQ2/8 (OR = 1.509; 95th CI 1.011, 2.252; P = 0.045) but not in 2/2, 2/X, 8/8, 8/X or X/X (X is neither 2 nor 8) patients. The association with HLA-DQ2/8 suggests that this autoantigen may be presented on HLA-DQ trans-heterodimers, rather than cis-heterodimers. Autoantibodies reactive with both insulin and INS-IGF2A at diagnosis support the notion that INS-IGF2 autoimmunity contributes to type 1 diabetes.

The three ZNT8 autoantibody variants together improve the diagnostic sensitivity of childhood and adolescent type 1 diabetes.

AIMS: We tested whether autoantibodies to all three ZnT8RWQ variants, GAD65, insulinoma-associated protein 2 (IA-2), insulin and autoantibodies to islet cell cytoplasm (ICA) in combination with human leukocyte antigen (HLA) would improve the diagnostic sensitivity of childhood type 1 diabetes by detecting the children who otherwise would have been autoantibody-negative. METHODS: A total of 686 patients diagnosed in 1996-2005 in Skåne were analyzed for all the seven autoantibodies [arginin 325 zinc transporter 8 autoantibody (ZnT8RA), tryptophan 325 zinc transporter 8 autoantibody (ZnT8WA), glutamine 325 Zinc transporter 8 autoantibody (ZnT8QA), autoantibodies to glutamic acid decarboxylase (GADA), Autoantibodies to islet-antigen-2 (IA-2A), insulin autoantibodies (IAA) and ICA] in addition to HLA-DQ genotypes. RESULTS: Zinc transporter 8 autoantibody to either one or all three amino acid variants at position 325 (ZnT8RWQA) was found in 65% (449/686) of the patients. The frequency was independent of age at diagnosis. The ZnT8RWQA reduced the frequency of autoantibody-negative patients from 7.5 to 5.4%-a reduction by 28%. Only 2 of 108 (2%) patients who are below 5 years of age had no autoantibody at diagnosis. Diagnosis without any islet autoantibody increased with increasing age at onset. DQA1-B1(*)X-0604 was associated with both ZnT8RA (p = 0.002) and ZnT8WA (p = 0.01) but not with ZnT8QA (p = 0.07). Kappa agreement analysis showed moderate (>0.40) to fair (>0.20) agreement between pairs of autoantibodies for all combinations of GADA, IA-2A, ZnT8RWQA and ICA but only slight ( < 0.19) agreement for any combination with IAA. CONCLUSIONS: This study revealed that (1) the ZnT8RWQA was common, independent of age; (2) multiple autoantibodies were common among the young; (3) DQA1-B1(*)X-0604 increased the risk for ZnT8RA and ZnT8WA; (4) agreement between autoantibody pairs was common for all combinations except IAA. These results suggest that ZnT8RWQA is a necessary complement to the classification and prediction of childhood type 1 diabetes as well as to randomize the subjects in the prevention and intervention of clinical trials.

Temporal trends of HLA genotype frequencies of type 1 diabetes patients in Sweden from 1986 to 2005 suggest altered risk.

The aim of this study was to compare the frequency of human leukocyte antigen (HLA) genotypes in 1-18-year-old patients with type 1 diabetes newly diagnosed in 1986-1987 (n = 430), 1996-2000 (n = 342) and in 2003-2005 (n = 171). We tested the hypothesis that the HLA DQ genotype distribution changes over time. Swedish type 1 diabetes patients and controls were typed for HLA using polymerase chain reaction amplification and allele specific probes for DQ A1* and B1* alleles. The most common type 1 diabetes HLA DQA1*-B1*genotype 0501-0201/0301-0302 was 36% (153/430) in 1986-1987 and 37% (127/342) in 1996-2000, but decreased to 19% (33/171) in 2003-2005 (P \ 0.0001). The 0501-0201/0501-0201 genotype increased from 1% in 1986-1987 to 7% in 1996-2000 (P = 0.0047) and to 5% in 2003-2005 (P > 0.05). This study in 1-18-year-old Swedish type 1 diabetes patients supports the notion that there is a temporal change in HLA risk.

Children developing type 1 diabetes before 6 years of age have increased linear growth independent of HLA genotypes.

AIMS/HYPOTHESIS: High birthweight and increased childhood growth are risk factors for type 1 diabetes. Relative birthweight is associated with HLA genotypes that confer a high risk of diabetes. Our aims were to test whether young children prior to clinical onset of type 1 diabetes have increased: (1) birthweight or birth length standard deviation scores (SDS); (2) height development SDS; or (3) BMI SDS during first 18 months of life and whether these parameters are related to HLA genotypes or mid-parental height (MPH). METHODS: Birthweight, birth length, weight and height were obtained from 58 type 1 diabetes children and 155 controls matched for HLA or not in the Diabetes Prediction in Skåne study. RESULTS: Birth length SDS corrected for MPH was increased in children developing diabetes compared with all (p < 0.048) and with non-HLA- (p < 0.050) but not with HLA-matched controls. Children developing diabetes had increased height gain at 0 to 18 months of age (p < 0.005). Diabetic children were significantly taller from 6 to 18 months of age when correcting for MPH compared with non-HLA-matched as well as HLA-matched controls, but BMI was not increased. CONCLUSIONS/INTERPRETATION: Birth length SDS was associated with diabetes risk HLA. When corrected for MPH, children developing diabetes were taller at birth than non-HLA- but not taller than HLA-matched controls. Diabetic children had increased MPH-corrected height up to 18 months of age compared with both HLA- and non-HLA-matched controls. High-risk HLA affects prenatal growth, but other factors may explain the increased postnatal linear growth in children developing diabetes.

IA-2 autoantibodies in incident type I diabetes patients are associated with a polyadenylation signal polymorphism in GIMAP5.

In a large case-control study of Swedish incident type I diabetes patients and controls, 0-34 years of age, we tested the hypothesis that the GIMAP5 gene, a key genetic factor for lymphopenia in spontaneous BioBreeding rat diabetes, is associated with type I diabetes; with islet autoantibodies in incident type I diabetes patients or with age at clinical onset in incident type I diabetes patients. Initial scans of allelic association were followed by more detailed logistic regression modeling that adjusted for known type I diabetes risk factors and potential confounding variables. The single nucleotide polymorphism (SNP) rs6598, located in a polyadenylation signal of GIMAP5, was associated with the presence of significant levels of IA-2 autoantibodies in the type I diabetes patients. Patients with the minor allele A of rs6598 had an increased prevalence of IA-2 autoantibody levels compared to patients without the minor allele (OR=2.2; Bonferroni-corrected P=0.003), after adjusting for age at clinical onset (P=8.0 x 10(-13)) and the numbers of HLA-DQ A1*0501-B1*0201 haplotypes (P=2.4 x 10(-5)) and DQ A1*0301-B1*0302 haplotypes (P=0.002). GIMAP5 polymorphism was not associated with type I diabetes or with GAD65 or insulin autoantibodies, ICA, or age at clinical onset in patients. These data suggest that the GIMAP5 gene is associated with islet autoimmunity in type I diabetes and add to recent findings implicating the same SNP in another autoimmune disease.

SUMO4 M55V polymorphism affects susceptibility to type I diabetes in HLA DR3- and DR4-positive Swedish patients.

SUMO4 M55V, located in IDDM5, has been a focus for debate because of its association to type I diabetes (TIDM) in Asians but not in Caucasians. The current study aims to test the significance of M55V association to TIDM in a large cohort of Swedish Caucasians, and to test whether M55V is associated in those carrying human leukocyte antigen (HLA) class II molecules. A total of 673 TIDM patients and 535 age- and sex-matched healthy controls were included in the study. PCR-RFLP was performed to identify the genotype and allele variations. Our data suggest that SUMO4 M55V is not associated with susceptibility to TIDM by itself. When we stratified our patients and controls based on heterozygosity for HLA-DR3/DR4 and SUMO4 genotypes, we found that presence of SUMO4 GG increased further the relative risk conferred by HLA-DR3/DR4 to TIDM, whereas SUMO4 AA decreased the risk. From the current study, we conclude that SUMO4 M55V is associated with TIDM in association with high-risk HLA-DR3 and DR4, but not by itself.

Thrombocytopenic purpura and anemia in a breast-fed infant whose mother was treated with valproic acid.

Valproic acid is an antiepileptic drug in widespread use. The possibility of various hematologic side effects with this drug is well recognized. We describe a breast-fed infant with thrombocytopenic purpura, anemia, and reticulocytosis, whose mother was treated with valproic acid. As the mother stopped breast-feeding, the infant recovered.

Prevalence of diabetic retinopathy in children and adolescents with IDDM. A population-based multicentre study.

Vision-threatening diabetic retinopathy can be prevented if it is diagnosed before becoming too advanced. Since diabetic retinopathy has been reported to occur only rarely before the end of pubertal development, children and adolescents are seldom included in screening programmes. We invited 780 children and adolescents with insulin-dependent diabetes mellitus diagnosed before the age of 15.0 years (disease duration of < 12 years) and who were older than 9.0 years at the time of examination from eight regions of Sweden. Retinal examination was performed with stereoscopic fundus photograph. The photograph were rated according to a modified Airlie House classification. The dropouts (223/780, 28.6%) were significantly older and with a longer duration of diabetes than the examined children (p < 0.001 and 0.001, respectively). Photographs from 557 patients aged (median [interquartile range]:14.6 [12.4-17.0]) years and with a diabetes duration of 8.0 (5.5-9.9) years were evaluated. Retinopathy was demonstrated in 81 patients (14.5%):66 with background retinopathy, 2 with microaneurysms and hard exudates, 12 with preproliferative retinopathy, 1 with proliferative retinopathy. Preproliferative retinopathy was diagnosed in a 12.8-year-old girl in pubertal stage 3 and an 11.8-year-old boy in pubertal stage 2, and proliferative retinopathy was found in a 21.5-year-old girl. Retinopathy was demonstrated in 6% and 18% of patients in pubertal stages 1 and 5, respectively. The overall prevalence of retinopathy in this population may even be higher since the dropouts were older and had a longer duration of diabetes. Since background and preproliferative retinopathy were found in children before puberty, we recommend including children and adolescents in screening programmes for diabetic retinopathy from the age of 10 years.

Munchausen syndrome by proxy: an unexpected cause of severe chronic diarrhoea in a child.

A six-year-old boy with severe intermittent diarrhoea was subjected to extensive diagnostic investigation and treatment before the administration of a laxative (sodium sulphate) by his mother was discovered. Early suspicion and analysis of faecal fluid for electrolytes and known laxative substances would have prevented the hazardous clinical course of this case.

Greek immigrant children in southern Sweden in comparison with Greek and Swedish children. III. Energy and nutrient intake.

Greek immigrant children (GI) belonging to the second generation of immigrants in Sweden have been compared with Swedish children (S) and Greek children in Greece (G) regarding energy and nutrient intake. Twenty-four-hour recalls were obtained in the homes of the families. The mean energy intake was the same in all three groups and met the Swedish Nutrition Recommendations. The energy distribution for protein, carbohydrates and fat was also similar. The fat intake was far above the recommended level in all groups. The GI and the G group had a significantly higher mean daily intake of monounsaturated fatty acids than the S group (p less than 0.05 and p less than 0.001, respectively). Children aged four to eight years in the GI group had a significantly higher nutrient density of retinol, vitamin D, riboflavin, vitamin B6, calcium, iron, magnesium and sucrose compared to the G group, but compared to the S group they had a lower nutrient density of retinol, vitamin D, ascorbic acid, niacin, vitamin B12 and selen. The GI children consumed more milk than the G children but as much as the S children and they had started to use enriched low-fat milk in Sweden. In conclusion, the food quality in the GI group was better than in the G group and much the same as in the S group, and, with few exceptions, it met the Swedish Nutrition Recommendations.

Dental health and dietary habits in Greek immigrant children in southern Sweden compared with Swedish and rural Greek children.

Dental health and dietary habits were surveyed in 40 Greek immigrant (GI) children, 2-8 years old, born and living in Helsingborg, Sweden; comparisons were made with 45 Swedish (S) and 54 rural Greek (G) children of the same age. The caries situation was virtually the same in the GI and the S group, where the primary teeth were caries-free in 31-33%, mainly children 2-3 years old. The G group had a higher incidence of decayed and filled tooth surfaces in both primary and permanent teeth than the other two groups and only 15% were caries-free in the primary teeth. The S group had the lowest gingival bleeding index. The distribution of the mutans streptococci and lactobacilli counts in saliva did not differ significantly between the three groups, except that the proportion of GI children with "not detectable" mutans streptococci was lower in either the S or the G group. The toothbrushing frequency was highest in the S group, followed by the GI group. Approximately 80% of the S children who brushed their teeth used a fluoride toothpaste compared to 50 and 55% respectively in the G and the GI group. The intake frequency for 5 out of 6 preselected snack-food items was highest in the G group. The carbohydrate content of the diet, including sucrose, was approximately the same in the three groups. Thus, the dental health and dietary habits of the Greek immigrant and the Swedish children were generally very similar, while the Greek rural children showed a less favourable cariological status.

Greek immigrant children in southern Sweden in comparison with Greek and Swedish children. II. Meal pattern and food habits.

Greek immigrant children (GI) belonging to the second generation of immigrants in Sweden have been compared with Swedish children (S) and Greek children in Greece (G) regarding meal pattern and food habits. Interviews were performed in the homes. Meal pattern and frequency of consumption of various food items were studied by 24-hour recalls, and the food frequency interview method. The meal pattern and the distribution of meals and snacks during the day did not show large differences between the groups. In the GI and G groups it was common to start the day with milk and sugar. The same habit was reported before going to bed. It was common in the GI and G groups to have a prepared meal (dinner) relatively late in the evening. Of the S children only 55% had dinner. GI children had a less frequent intake of vegetables compared to S and G children. GI and G children were heavy consumers of sweets and snacks. In conclusion, the Greek immigrant group had food habits similar to those of Greek rural children with few distinctive exceptions. Greek immigrant families had no difficulties in preparing Greek food.

Greek immigrant children in southern Sweden in comparison with Greek and Swedish children. I. General living conditions.

Greek immigrant children belonging to the second generation of immigrants in Sweden have been compared with Swedish children and Greek children in Greece regarding general living conditions. Interviews were performed in the homes of all participants. The parents in the two Greek groups had the lowest educational level. The yearly salary of immigrant families was similar to that of the Swedish families. Immigrant and Swedish parents worked outside their homes to the same extent, Greek immigrant mothers fulltime, Swedish mothers mostly part time. For economical reasons the immigrant parents looked after their children within the family while the Swedish families almost always utilized community day care facilities. The immigrant families had fewer children than the Swedish and Greek rural families and their dwellings were smaller. Corporal punishment was a common method of upbringing in Greece and among the immigrants. The immigrant families had extremely few contacts with Swedish families. A majority of the immigrant families were unsure about their future in Sweden, whether or not to stay. In conclusion, the Greek immigrant group in many respects had adapted to Swedish customs but they had also at the same time retained much of the Greek cultural characteristics.