Seasonal variation in month of diagnosis in children with type 1 diabetes registered in 23 European centers during 1989-2008: little short-term influence of sunshine hours or average temperature.

BACKGROUND: The month of diagnosis in childhood type 1 diabetes shows seasonal variation. OBJECTIVE: We describe the pattern and investigate if year-to-year irregularities are associated with meteorological factors using data from 50 000 children diagnosed under the age of 15 yr in 23 population-based European registries during 1989-2008. METHODS: Tests for seasonal variation in monthly counts aggregated over the 20 yr period were performed. Time series regression was used to investigate if sunshine hour and average temperature data were predictive of the 240 monthly diagnosis counts after taking account of seasonality and long term trends. RESULTS: Significant sinusoidal pattern was evident in all but two small centers with peaks in November to February and relative amplitudes ranging from ± 11 to ± 38% (median ± 17%). However, most centers showed significant departures from a sinusoidal pattern. Pooling results over centers, there was significant seasonal variation in each age-group at diagnosis, with least seasonal variation in those under 5 yr. Boys showed greater seasonal variation than girls, particularly those aged 10-14 yr. There were no differences in seasonal pattern between four 5-yr sub-periods. Departures from the sinusoidal trend in monthly diagnoses in the period were significantly associated with deviations from the norm in average temperature (0.8% reduction in diagnoses per 1 °C excess) but not with sunshine hours. CONCLUSIONS: Seasonality was consistently apparent throughout the period in all age-groups and both sexes, but girls and the under 5 s showed less marked variation. Neither sunshine hour nor average temperature data contributed in any substantial way to explaining departures from the sinusoidal pattern.

Trends in childhood type 1 diabetes incidence in Europe during 1989-2008: evidence of non-uniformity over time in rates of increase.

AIMS/HYPOTHESIS: The aim of the study was to describe 20-year incidence trends for childhood type 1 diabetes in 23 EURODIAB centres and compare rates of increase in the first (1989-1998) and second (1999-2008) halves of the period. METHODS: All registers operate in geographically defined regions and are based on a clinical diagnosis. Completeness of registration is assessed by capture-recapture methodology. Twenty-three centres in 19 countries registered 49,969 new cases of type 1 diabetes in individuals diagnosed before their 15th birthday during the period studied. RESULTS: Ascertainment exceeded 90% in most registers. During the 20-year period, all but one register showed statistically significant changes in incidence, with rates universally increasing. When estimated separately for the first and second halves of the period, the median rates of increase were similar: 3.4% per annum and 3.3% per annum, respectively. However, rates of increase differed significantly between the first half and the second half for nine of the 21 registers with adequate coverage of both periods; five registers showed significantly higher rates of increase in the first half, and four significantly higher rates in the second half. CONCLUSIONS/INTERPRETATION: The incidence rate of childhood type 1 diabetes continues to rise across Europe by an average of approximately 3-4% per annum, but the increase is not necessarily uniform, showing periods of less rapid and more rapid increase in incidence in some registers. This pattern of change suggests that important risk exposures differ over time in different European countries. Further time trend analysis and comparison of the patterns in defined regions is warranted.

Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia.

OBJECTIVE: To analyze the mutational spectrum of steroid 21-hydroxylase (CYP21) and the genotype- phenotype correlation in patients with congenital adrenal hyperplasia (CAH) registered in the Middle European Society for Pediatric Endocrinology CAH database, and to design a reliable and rational approach for CYP21 mutation detection in Middle European populations. DESIGN AND METHODS: Molecular analysis of the CYP21 gene was performed in 432 CAH patients and 298 family members. Low-resolution genotyping was performed to detect the eight most common point mutations. High-resolution genotyping, including Southern blotting and sequencing was performed to detect CYP21 gene deletions, conversions, point mutations or other sequence changes. RESULTS: CYP21 gene deletion and In2 and Ile172Asn mutation accounted for 72.7% of the affected alleles in the whole study group. A good genotype-phenotype correlation was observed, with the exception of Ile172Asn and Pro30Leu mutations. In 37% of patients low resolution genotyping could not identify the causative mutation or distinguish homozygosity from hemizygosity. Using high-resolution genotyping, the causative mutations could be identified in 341 out of 348 analyzed patients. A novel mutation Gln315Stop was found in one simple virilising CAH (SV-CAH) patient from Austria. In the remaining seven patients polymorphisms were identified as the leading sequence alteration. The presence of elevated basal and ACTH-stimulated 17-hydroxyprogesterone, premature pubarche, advanced bone age and clitoral hypertrophy directly implicated Asn493Ser polymorphism in the manifestation of nonclassical- (NC) and even SV-CAH. CONCLUSIONS: By genotyping for the most common point mutations, CYP21 gene deletion/conversion and the 8 bp deletion in exon 3, it should be possible to identify the mutation in 94-99% of the diseased alleles in any investigated Middle European population. In patients with a mild form of the disease and no detectable mutation CYP21 gene polymorphisms should be considered as a plausible disease-causing mutation.

Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease.

OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

Tissue glucose transport and glucose transporters in suckling rats with endotoxic shock.

Hypoglycemia occurs without hyperinsulinemia in suckling rats with endotoxic shock. However, tissue glucose uptake during endotoxic shock is not well known in the newborn. GLUT1 is insulin insensitive and is the predominant glucose transporter in 10 day old rats. In the adult with endotoxic shock, noninsulin-mediated glucose uptake and GLUT1 gene expression increase. Therefore, we hypothesized that tissue glucose uptake and GLUT1 mRNA abundance increased in 10 day old rats with endotoxic shock. The present study showed that whole body glucose disposal increased 3 h after a Salmonella enteritidis lipopolysaccharide injection (LD90 at 72 h). Plasma insulin concentration was not altered. Tissue glucose uptake increased in liver (2.4-fold) and fat (2.6-fold). However, changes of GLUT1 protein concentration were not detected in liver. GLUT1 mRNA abundance increased in liver (9-fold) and fat (4-fold). GLUT1 mRNA abundance but not glucose uptake increased in muscle. Neither glucose uptake or GLUT1 mRNA abundance was altered in brain. The mRNA abundance of tissue-specific glucose transporters (GLUT2 and GLUT4) was not altered. Thus, tissue glucose uptake and GLUT1 mRNA abundance increased without hyperinsulinemia during endotoxic shock in 10 day old rats.

Incidence of type 1 diabetes mellitus in children in Slovenia during the years 1988-1995.

The incidence of type 1 diabetes mellitus in Slovenian children aged 0-14 years was studied between 1 January 1988 and 31 December 1995. The crude annual incidence rate of the disease (per 100000) over this 8-year period was 8.00 (95% C. I. 6.98-9.02) for both sexes (7.18 for boys and 8.87 for girls). Thus, the incidence standardized to the world population was 7.59 (95% C. I. 6.57 - 8.61). Male/female ratios were 1.33 in the age group 0-4 years, 0.66 in the age group 5-9 years, and 0.83 in the age group 10-14 years. The study has proven that the incidence of type 1 diabetes in Slovenia is similar to that in other central European countries where the population is of different ethnic origin. However, a remarkably higher incidence of the disease in girls than boys except in the age group below 5 years of age was found which needs further investigation.

Motor activity during asymptomatic nocturnal hypoglycemia in adolescents with type 1 diabetes mellitus.

Nocturnal hypoglycemia is reported in 13%-56% of adolescents with type 1 diabetes mellitus. It may be asymptomatic in more than 50% of patients. No noninvasive method for detecting asymptomatic nocturnal hypoglycemia (ANH) has so far proven successful. The aim of the present study was to evaluate quantitative changes of motor activity by actigraphy during episodes of ANH in adolescents with type 1 diabetes mellitus. A total of 18 patients aged 10-16 years with a history of ANH were investigated. Blood was sampled at half-hourly intervals between 22.30 and 06.00 hours with a micropump, and an actigraph was fastened to the right wrist. Blood glucose concentrations were measured and compared to motor activity. Nocturnal hypoglycemia was recorded in 10 patients (55%), with blood glucose during periods of hypoglycemia of 3.00+0.17 mmol/l (range, 1.2-3.4 mmol/l), and duration of hypoglycemia of 1.95+1.34 hours (range, 0.5-5.0 hours). All periods of hypoglycemia were clinically asymptomatic. Regression analysis revealed a statistically significant linear correlation ( p=0.03) between blood glucose concentration and the respective 30-min activity counts. Activity counts in patients with nocturnal hypoglycemia were significantly (ANOVA, p<0.02) higher than in patients with normoglycemia. We conclude that low blood glucose was significantly correlated with an increase in motor activity as detected by actigraphy. This implies the possibility of noninvasive screening of asymptomatic nocturnal hypoglycemia.

Frequency of Hashimoto's thyroiditis in children with type 1 diabetes mellitus.

A total of 1419 children with type 1 diabetes mellitus was investigated in order to assess the true frequency of Hashimoto's thyroiditis (HT), diagnosed by microsomal and/or thyroglobulin autoantibodies, by ultrasound and in many cases also by fine needle biopsy. According to these criteria, 55 cases (3.9%) of HT were identified, a number significantly higher (P < 0.0001) than the distribution reported in the normal paediatric population. No typical antibody pattern was seen prior to the onset of HT, nor was an antibody threshold level found which could have been diagnostic for this disease. Patients with subclinical hypothyroidism were treated with L-thyroxine and were investigated regarding the behaviour of anti-thyroid autoantibodies; however, no significant changes were seen. The data showed a high frequency of HT in diabetic children, and therefore we recommend that children with type 1 diabetes mellitus should be screened for thyroid autoantibodies and those positive should undergo periodic thyroid function testing.

Testing for thyroid function recovery in children and adolescents with Hashimoto thyroiditis.

Thyroid function in children and adolescents with primary hypothyroidism owing to Hashimoto thyroiditis was studied to evaluate criteria for the discontinuation of thyroxine therapy. A cohort of 29 children and adolescents was prospectively studied for one year. A thyrotropin-releasing hormone stimulation test with measurements of basal and stimulated thyrotropin and thyroxine was performed at the beginning of the study and 6 and 12 months later. In 59 percent of patients, persistent biochemical evidence of hypothyroidism was observed. Interindividual variations and variations among measurements in individual patients were much greater than those reported for healthy individuals. In one patient, all measured values were consistently normal, and the therapy was successfully discontinued. No single measurement or test could predict the natural course of disease. In summary, children and adolescents with hypothyroidism owing to Hashimoto thyroiditis can not have discontinued replacement thyroxine therapy on the basis of any single evaluation of thyroid function, as proposed for adults. If the decision to discontinue the therapy in this age group is made on the basis of previous follow-up, a substantial possibility of relapse remains and continuous follow up is necessary.

Five year treatment with IGF-I of a patient with Laron syndrome in Slovenia (a follow-up report).

This is a follow-up report of a 14 1/2 year-old boy with Laron syndrome, who received twice daily therapy with IGF-I 120 micrograms/kg for 5 years that resulted in a linear growth of 40 cm. Concomitantly he became very obese which is attributed to IGF-I action via the insulin receptors.

Seasonality of birth in children (0-14 years) with type 1 diabetes mellitus in Slovenia.

The aim of this study was to find out whether there is seasonality of month of birth of children with diabetes in Slovenia and if so whether it differs from that of the general population. A cohort of 849 children and adolescents (0-14 years) with type 1 diabetes mellitus born between 1956 and 1998 were included in the study. Monthly and seasonal patterns of birth of the patients with diabetes were compared with the pattern of normal live births (n = 1,345,921) and the pattern of disease onset. Statistical analysis was made using Student's t-test to compare the means between the four seasons of the year, and single cosinor analysis for a period of 12 months. The children and adolescents with diabetes had a statistically significant different seasonality of month of birth compared to that of the general population, and an opposite pattern from the seasonality of month of onset of disease. The observations made are in accordance with observations made recently in other countries and support the hypothesis that a virus infection transmitted by the mother to the fetus during the annual viral epidemic induces the autoimmune process in the pancreatic beta-cells in genetically susceptible individuals who will subsequently develop clinical diabetes during childhood.

Goiter prevalence and urinary iodine concentration in Slovenian adolescents.

CONTEXT: Slovenian school-age children are, as are more than half of European school-age children, still considered to be iodine deficient. In 1999, supplementation of salt was increased from 10 to 25 mg of KI/kg of salt. OBJECTIVE: The objective of our study was to determine the success of this intervention. DESIGN AND PATIENTS: Twelve hundred sixty-four girls (mean age +/- SD: 15.7 +/- 0.6 years) and 1200 boys (15.8 +/- 0.8 years) representing 10% of all 15-year-old Slovenian adolescents were studied. Thyroid size was estimated by clinical examination in all subjects and by ultrasound when enlarged thyroid was suspected. Thyroid volume was also determined by ultrasound in 108 random iodine-sufficient adolescents. In addition, urinary iodine concentration was determined in all subjects. RESULTS: Enlarged thyroid was determined by clinical examination and ultrasound in 0.9% of all subjects. In randomly selected iodine-sufficient subjects, enlarged thyroid was determined in 4.6%. Median urinary iodine concentration for the population was 140 microg/L. In all regions it was greater than or equal to 100 microg/L. Values less than 50 microg/L were determined in 2.5% of all subjects. CONCLUSIONS: Slovenian adolescents are iodine sufficient and the prevalence of goiter is low, indicating that increased KI supplementation of salt in 1999 was successful.

Glycosylated hemoglobin in fractions of erythrocytes of different ages.

In 65 children and adolescents with type I diabetes and in 23 healthy controls, glycosylated hemoglobin (HbA1) was measured by ion exchange chromatography in a specimen of whole blood and in three fractions of erythrocytes of different ages. The red blood cells were separated in a density-gradient medium. Three layers were obtained. The top one consisted of younger and lighter cells, while the heaviest and oldest cells were found in the bottom layer. The separation was evaluated by measuring the potassium concentration, which was significantly different in each fraction (p < 0.001). In all healthy controls the lowest HbA1 value was measured in the youngest and the highest in the oldest erythrocytes. In the diabetic patients, however, the HbA1 levels in individual red cell fractions depended on the degree of metabolic control during the life span of the cells. Significant correlation was found between the HbA1 levels in the youngest erythrocytes and metabolic control in the two-week period before investigation (p < 0.001), between the HbA1 content of the medium-aged cell fraction and control two months before assay (p < 0.001), and between HbA1 levels in the oldest erythrocytes and control three and especially four months prior to investigation (p < 0.001). The results of HbA1 determination in the fraction of the youngest cells which show the highest correlation (-0.75) with the glucosuria two weeks prior to assay indicate to be one of possibilities to get information about metabolic control in that period.

A case of Laron syndrome diagnosed in Slovenia.

We report the first case of Laron syndrome (LS) diagnosed in Slovenia. The boy, a product of non-consanguineous Slovenian parents of normal height, presented with slow growth and motor development since birth. At age 4 and 6 years, he had all the characteristic signs of LS, identical to those in growth hormone deficiency (GHD). Laboratory tests showed hypoglycemia, markedly elevated plasma hGH, low serum insulin-like growth factor-1 (IGF-1) with no rise after exogenous hGH, and low serum growth hormone binding protein (GHBP). A sister of the maternal grandfather is short (145 cm) and was found to have below normal serum GHBP, findings compatible with heterozygocity for this disorder.

Incidence of childhood-onset Type I diabetes in Slovenia and the Tuzia region (Bosnia and Herzegovina) in the period 1990-1998.

AIMS/HYPOTHESIS: The incidence rate of childhood-onset Type I (insulin-dependent) diabetes mellitus in Slovenian children during the period 1990-1998 was studied and compared to that of children from the Tuzla region (Bosnia and Herzegovina). The secular trend for a 25-year period in Slovenia was also investigated. METHODS: The incidence data were obtained from the national Slovenian Type I diabetes register and from the local Type I diabetes register held in Tuzla. The ascertainment was based on the capture-recapture method and was estimated to be 100%. RESULTS: The age-standardized incidence of Type I diabetes mellitus for the age group 0-14 years for Slovenia was 8.54 per 100,000 (95 %-C.I. 7.5-9.5) person-years for both sexes. The incidence for boys was 8.03 per 100,000 (95 %-C.I. 6.7-9.4) and 9.12 per 100,000 (95% -C.I. 7.6-10.6) for girls. The age-standardized incidence in the Tuzla region was much lower: 3.03 per 100,000 (95 %-C.I. 2.0-4.1) for the whole group, 3.44 per 100 000 (95 %-C.I. 1.8-5.0) for boys and 3.21 per 100,000 (95 %-C.I. 1.6-4.7) for girls. A very low incidence of 0.8 per 100,000 in the youngest age group (0-4 years) was observed in the Tuzla region. The linear trend of the Type I diabetes incidence rate in Slovenia has been steadily increasing by 3.6% per year for the last 25 years. The incidence rate in the period 1974-1985 was significantly lower than that in the period 1986-1998 (p < 0.00026). CONCLUSIONS/INTERPRETATION: Although the incidence rate in Slovenia slightly increased during the period 1990-1998, the incidence rate in the Tuzla region remained at the pre-war level.

Tumor necrosis factor-alpha alters glucose metabolism in suckling rats.

Tumor necrosis factor-alpha (TNF-alpha), an important mediator of endotoxic shock, induces hypoglycemia and shock in adult animals. Indomethacin ameliorates TNF-alpha-induced hypoglycemia in the adult. However, effects of TNF-alpha on glucose metabolism in the newborn have not been well documented. The present study showed that in 10-day-old rats injected with TNF-alpha (4.5 x 10(7) U/kg, intraperitoneally) the plasma glucose concentration increased from 4.1 +/- 0.3 mmol/L to 6.9 +/- 0.5 mmol/L (P < .05) at 2 hours and subsequently decreased to 1.4 +/- 0.5 mmol/L (P < .05) at 6 hours, although plasma lactate concentration increased from 1.1 +/- 0.1 mmol/L to 5.5 +/- 0.3 mmol/L (P < .05) at 6 hours. Plasma insulin concentration remained unchanged throughout the experiment. TNF-alpha increased GLUT 1 messenger RNA (mRNA) abundance in the brain, liver, muscle, and fatty tissue (P < .05). Glucose uptake increased in association with the increase of GLUT1 mRNA abundance. TNF-alpha decreased mRNA abundance of GLUT 2 and phosphoenolpyruvate carboxykinase (PEPCK) in liver, suggesting decreased gluconeogenesis. Indomethacin (1.5 mg/kg 20 minutes before TNF-alpha, intraperitoneally) attenuated the hypoglycemia, the lactacidemia, and the increase of GLUT1 mRNA abundance and glucose uptake. Indomethacin attenuated the decrease of PEPCK mRNA abundance. We concluded that TNF-alpha induced hypoglycemia, increasing GLUT1 mRNA abundance and glucose uptake and decreasing PEPCK mRNA abundance in 10-day-old rats. Indomethacin attenuated the TNF-alpha-induced glucose dyshomeostasis.

TGF-beta activates genes identified by differential mRNA display in pancreatic rudiments.

The effect of TGF-beta on gene activation in embryonic pancreatic rudiments was investigated using differential mRNA display. Several cDNA bands were augmented and some were suppressed in the presence of TGF-beta. Differentially expressed cDNAs were re-amplified, sequenced, and sequences compared to the GeneBank database. Glucagone and brain alpha-tropomyosin cDNAs were identified from the group of augmented cDNAs, and B-carboxypeptidase form the group of suppressed cDNAs. PCR experiments were confirmed with Northern blots. Obtained results are in accordance with immunohistochemical findings and render differential mRNA display a useful technique in identifying differentially expressed genes in embryonic pancreatic rudiments. Several unknown differentially expressed cDNA sequences obtained in our experiments remain to be identified.

Late sequelae in children treated for brain tumors and leukemia.

Forty-two survivors treated at an age of 2-16 years for brain tumors or leukemia were, 4-21 years after treatment, subjected to an extensive follow-up investigation, including physical examination and interview; 35 of them also had endocrinological and 33 psychological evaluation. Hormonal deficiencies were found in about two-thirds of patients and were most common in those treated for brain tumors. The great majority had verbal intelligence quotient (VIQ) within normal range. Also, the performance intelligence quotients (PIQ) were normal in most patients. However, the results suggested that the primary intellectual capacity in children treated for cancer was not being fully utilized, their PIQ being on the average higher than their VIQ; this tendency was especially pronounced in the leukemia patients.

Similar incidence of type 1 diabetes in two ethnically different populations (Italy and Slovenia) is sustained by similar HLA susceptible/protective haplotype frequencies.

The incidence of type 1 diabetes (T1DM) seems to depend in part on the population frequencies of susceptible and protective HLA haplotypes. The present study aimed to (i): characterize the genetic susceptibility to T1DM in the Slovenian population, (ii) test the general hypothesis that T1DM incidence is related to the frequencies of susceptible/protective haplotypes, (iii) compare allele, haplotype and genotype frequencies in Slovenians and Italians that represent two white populations with a similar incidence of T1DM (7.9/100,000/year and 8.1/100,000/year, respectively). The haplotype found most frequently among Slovenian T1DM patients was DRB1*0301-DQA1*0501-DQB1*0201 (53%). The DR4-DQA1*0301-DQB1*0302 haplotypes conferring susceptibility to T1DM were those bearing DRB1*0401 (OR = 12), DRB1*0404 (OR = 4.7) and DRB1*0402 (OR = 4.5). Negative associations with the disease were found for the following haplotypes: DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1301-DQA1*0102-DQB1*0603, DRB1*1101/1104-DQA1*0501-DQB1*0301, and DRB1*1401-DQA1*0101-DQB1*0503. Our findings indicate that the low frequencies of susceptible genotypes, in particular, DR3-DQA1*0501-DQB1*0201/DR4-DQA1*0301-DQB1*0302, together with a high frequency of protective haplotypes, could in part explain the low incidence of T1DM in the Slovenian population. The combined frequencies of susceptible genotypes were similar in the two populations (Slovenia = 19.2%, Italy = 17.6%), and the 95% confidence limits of the OR values for each genotype in the two populations overlapped, indicating no significant differences between the values. We conclude that the similar incidences of T1DM in Italian and Slovenian populations are in part a reflection of similar frequencies of HLA susceptible/protective haplotypes.