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.

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.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro.

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFalpha, TGFbeta and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned gamma-chain of human or sheep fetal hemoglobin, but not cloned alpha- or beta-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, gamma-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.

Characterization of an interaction between fetal hemoglobin and lipid A of LPS resulting in augmented induction of cytokine production in vivo and in vitro.

A previously described extract of sheep fetal liver was reported to reverse many of the cytokine changes associated with aging in mice, including an augmented spleen cell ConA-stimulated production of IL-4 and decreased production of IL-2. Similar effects were not seen with adult liver preparations. These changes were observed in various strains of mice, including BALB/c, DBA/2 and C57BL/6, using mice with ages ranging from 8 to 110 weeks. Preliminary characterization of this crude extract showed evidence for the presence of Hb gamma chain, as well as of lipid A of LPS. We show below that purified preparations of sheep fetal Hb, but not adult Hb, in concert with suboptimally stimulating doses of LPS (lipid A), cooperate in the regulation of production of a number of cytokines, including TNFalpha and IL-6, in vitro. Furthermore, isolated fresh spleen or peritoneal cells from animals treated in vivo with the same combination of Hb and LPS, showed an augmented capacity to produce these cytokines on further culture in vitro. Evidence was also obtained for a further interaction between CLP, LPS and fetal Hb itself in this augmented cytokine production. These data suggest that some of the functional activities in the fetal liver extract reported earlier can be explained in terms of a novel immunomodulatory role of a mixture of LPS (lipid A) and fetal Hb.

Final height after combined growth hormone and GnRH analogue treatment in adopted girls with early puberty.

BACKGROUND: Girls adopted from developing countries often have early or precocious puberty, requiring treatment with gonadotrophin-releasing hormone (GnRH) analogues. During such treatment, decreased growth velocity is frequent. AIM: To study whether the addition of growth hormone (GH) to GnRH analogue treatment improves final height in girls with early or precocious puberty. METHODS: Forty-six girls with early or precocious puberty (age < or =9.5 y) adopted from developing countries were randomized for treatment for 2-4 y with GnRH analogue, or with a combination of GH and GnRH analogue. RESULTS: During treatment, the mean growth velocity in the GH/GnRH analogue group was significantly higher compared to the control group. Combined GH/GnRH analogue treatment resulted in a higher final height: 158.9 cm compared to 155.8 cm in the GnRH analogue-treated group. Three out of 24 girls (13%) in the combined group and nine of the 22 girls (41%) treated with GnRH analogue alone attained a final height below -2 standard deviation scores (SDS). CONCLUSION: The difference between the two groups is statistically significant, and possibly of clinical importance. A future challenge is to identify a subgroup with clinically significant advantage of GH addition to GnRH analogue treatment. Being very short on arrival in Sweden and being short and young at start of treatment are possible indicators.

Analysis of short stature homeobox-containing gene ( SHOX) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity.

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.

Mutations in short stature homeobox containing gene (SHOX) in dyschondrosteosis but not in hypochondroplasia.

Dyschondrosteosis (DCO) and hypochondroplasia (HCH) are common skeletal dysplasias characterized by disproportionate short stature. The diagnosis of these conditions might be difficult to establish especially in early childhood. Point mutations and deletions of the short stature homeobox containing gene (SHOX) are detected in DCO and idiopathic short stature with some rhizomelic body disproportion, whereas mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are found in 40-70% of HCH cases. In this study, we performed mutational analysis of the coding region of the SHOX gene in five DCO and 18 HCH patients, all of whom tested negative for the known HCH-associated FGFR3 mutations. The polymorphic CA-repeat analysis, direct sequencing and Southern blotting were used for detection of deletions and point mutations. The auxological and radiological phenotype of these patients was carefully determined. Three novel mutations in DCO patients were found: (1) a deletion of one base (de1272G) (according to GenBank accession nos. Y11536, Y11535), resulting in a premature stop codon at position 75 of the amino acid sequence; (2) the transversion C485G resulting in the substitution Leu132Val; and (3) the transversion G549T causing an Arg153Leu substitution. These substitutions segregate with the DCO phenotype and affect evolutionarily conserved homeodomain residues, based on a comparison of homeobox containing proteins in 13 species. Moreover, these changes were not found in 80 unrelated, unaffected individuals. This strongly suggests that these mutations are pathogenic. The phenotype of our patients with DCO and HCH varied from mild to severe shortness and body disproportion. These results further support clinical and genetic heterogeneity of dyschondrosteosis and hypochondroplasia.

Children with septo-optic dysplasia - how to improve and sharpen the diagnosis.

The data in this article are based on investigations performed in 25 children with suspected septo-optic dysplasia (SOD). There are many signs and methods that help in the diagnosis of SOD. In particular, the ocular fundus, abnormalities of the hypothalamo-pituitary axis and other midline brain structures should be described. In order to achieve a more holistic and functional diagnosis, the degree of neurological, neuropsychiatric and psychological involvement should also be stated. It has been suggested that SOD is associated with autosomal recessive inheritance, and it can be speculated that it is the result of genetic and environmental influences early in gestation. An early diagnosis can favourably influence the outcome of the affected child.

Prevalence of coeliac disease in Turner syndrome.

This study was undertaken to investigate the prevalence of coeliac disease in children and adolescents with Turner syndrome. Eighty-seven children and adolescents with Turner syndrome were screened for IgA-antiendomysium antibodies (EMA) and IgA-antigliadin antibodies (AGA), 5% (4/87) being found to be EMA-positive, and 15% (13/87) to have AGA levels above normal. Of the 10 patients who were either AGA- or EMA-positive and further investigated with intestinal biopsy, four manifested villous atrophy (i.e. all three of the EMA-positive patients, but only one of the seven AGA-positive patients). The results suggest EMA-positivity to be a good immunological marker for use in screening for coeliac disease, and such screening to be justified in patients with Turner syndrome.

Effect of growth hormone (GH) during puberty in GH-deficient children: preliminary results from an ongoing randomized trial with different dose regimens.

This paper reports results from an ongoing, randomized, multicentre national trial. The aim is to elucidate whether a dose of growth hormone (GH) of 0.2 IU/kg (0.07 mg/kg), given either as once-daily or twice-daily injections during puberty, is more effective than a once-daily dose of 0.1 IU/kg/day (0.03 mg/kg/day) in improving final height in children with GH deficiency (GHD). The twice-daily regimen comes closer to the spontaneous GH secretion pattern in puberty. Ninety-two children with GHD who had been receiving GH therapy for at least 1 year, and with spontaneous puberty or who were prepubertal and due to be started on replacement therapy to induce puberty, were randomly assigned to receive GH as follows: group A, 0.1 IU/kg/day (0.03 mg/kg/day), administered once daily; group B, 0.2 IU/kg/day (0.07 mg/kg/day), administered once daily; and group C, 0.2 IU/kg/day (0.07 mg/kg/day), divided into two equal injections given at 12-hour intervals. Pubertal height gain was 0.7, 0.7 and 1.3 SDS for groups A, B and C, respectively. The gain in height during puberty was thus most marked in group C. Mean final height, when corrected for parental height, was between 0 and 1 SDS in all treatment groups. All but seven children reached a final height within +/- 2 SD of the general population. There was a wide range of final heights in all three treatment groups. This variation in response suggests the need to individualize treatment in order to achieve an appropriate final height for most individuals.

A fetal sheep liver extract reverses age-related increments in spontaneous and induced cytokine production by indirect environmental effects.

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4-5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minimal changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.

Growth hormone treatment of short children born small-for-gestational-age: the Nordic Multicentre Trial.

The aims of this study were to evaluate the efficacy and safety of different doses of growth hormone (GH) treatment in prepubertal short children born small-for-gestational-age (SGA). Forty-eight children born SGA from Sweden, Finland, Denmark and Norway were randomly allocated to three groups: a control group of 12 children received no treatment for 2 y, one group was treated with GH at 0.1 IU/kg/d (n=16), and one group was treated with GH at 0.2 IU/kg/d (n=20). In total 42 children completed 2 y of follow-up, and 24 children from the treated groups completed 3 y of treatment. Their mean (SD) age at the start of the study was 4.69 (1.61) y and their mean (SD) height was -3.16 (0.70) standard deviation scores (SDS). The children remained prepubertal during the course of the study. No catch-up growth was observed in the untreated group, but a clear dose-dependent growth response was found in the treated children. After the third year of treatment, the group receiving the higher dose of GH, achieved their target height. The major determinants of the growth response were the dose of GH used, the age at the start of treatment (the younger the child, the better the growth response) and the family-corrected individual height deficit (the higher the deficit, the better the growth response). Concentration of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 increased during treatment. An increase in insulin levels was found without negative effects on fasting glucose levels or glycosylated haemoglobin levels. GH treatment was well tolerated. In conclusion, short prepubertal children born SGA show a dose-dependent growth response to GH therapy, and their target height SDS can be achieved within 3 y of treatment given GH at 0.2 IU/kg/d. However, the long-term benefit of different regimens of GH treatment in children born SGA remains to be established.

Radioiodine treatment of hyperthyroidism in a pregnant women.

UNLABELLED: We describe the effects of radioiodine treatment of a pregnant thyrotoxic woman. METHODS: The woman received 500 MBq of (131)I in her 20th gestational week. The pregnancy was discovered 10 days after radioiodine administration. A gamma camera examination of the abdomen at that time showed a distinct focus of activity, which was interpreted as the fetal thyroid. Gamma camera examinations of the mother and fetus were performed at 10, 11, 12, 13 and 18 days after administration of the therapeutic activity and were the basis of dose calculations. The child was examined by hormone tests and mental performance tests, up to 8 yr after birth. RESULTS: The uptake at 24 hr postadministration was calculated to be 10 MBq (2%) in the fetal thyroid gland. The effective half-life was 2.5 days, giving a calculated absorbed dose to the fetal thyroid gland of 600 Gy, which is considered to be an ablative dose. The calculated absorbed dose to the fetal body, including brain, was about 100 mGy, and 40 mGy to the fetal gonads. Doses were estimated taking contributions from radioiodine in the mother, the fetal body and the fetal thyroid into consideration. The woman was encouraged to continue her pregnancy and received levothyroxine in a dose to render her slightly thyrotoxic. At full term, an apparently healthy boy, having markedly raised cord blood serum thyroid-stimulating hormone concentration and subnormal thyroxine (T4) and low-normal triiodothyronine (T3) concentrations, was born. Treatment with thyroxine was initiated from the age of 14 days, when the somatosensoric evoked potential latency time increased to a pathological value and hormonal laboratory tests repeatedly confirmed the hypothyroid state. At 8 yr of age, the child attends regular school. A neuropsychological pediatric examination showed that the mental performance was within normal limits, but with an uneven profile. He has a low attention score and displays evidently subnormal capacity regarding figurative memory. CONCLUSION: Radioiodine treatment in pregnancy in the 20th gestational week does not give a total absorbed dose to the fetal body that justifies termination of pregnancy. A high absorbed dose to the fetal thyroid, however, should be the basis of the management of the pregnancy and offspring.

An antibody specific for interleukin-6 reverses age-associated changes in spontaneous and induced cytokine production in mice.

A number of quantitative and qualitative changes in the pattern of cytokine production have been reported to accompany the process of ageing in laboratory animals and in human populations, including an increase in serum levels of interleukin-1 (IL-1) and IL-6, as well as increased concanavalin A (ConA)-stimulated production of IL-4, IL-10 and transforming growth factor-beta (TGF-beta), and decreased production of IL-2 from cultured spleen cells. Increased IL-1 and IL-6 production is a feature of splenic adherent cells and peritoneal exudate cells taken from aged mice and stimulated with lipopolysaccharide in vitro. We have asked whether the altered production of lymphocyte-derived cytokines (IL-4, IL-2, TGF-beta) is itself a function of a primary alteration in IL-1/IL-6 production (from macrophage/monocytes) by infusing monoclonal antibodies to these cytokines prior to harvesting cells from aged mice and stimulating the cells in vitro. Anti-IL-6, but not anti-IL-1, reversed the age-associated alteration in lymphocyte cytokine production. The general pattern of cytokine production in aged mice is of a type-2 cytokine type, and thus these data are consistent with the idea that increased production of IL-6 in aged animals is causally implicated in this age-associated polarization to type-2 cytokine production.

Obese (ob) gene defects are rare in human obesity.

Our knowledge of the role of the recently cloned ob-protein (leptin) in the regulation of body fat stores is largely derived from experiments performed in mice. Different mouse models exhibit abnormalities in ob-gene expression, with extreme overexpression in mice which lack bioactive ob-protein, have nonfunctional ob-receptors or hypothalamic lesions, and undetectable expression in mice with suggested defects in regulatory elements. The aim of this study is to examine if defects, corresponding to those in mice, exist in human obesity. Adipose tissue was obtained from 94 adult obese subjects and from six children who had developed obesity after surgery in the hypothalamic region. Total RNA was isolated and ob-gene expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot. The coding region of the ob-gene was sequenced in both directions in the 94 obese adults. No mutations were detected in the coding region of the ob-gene and ob-gene expression was detectable in all subjects and none of the subjects had an extreme overexpression. There was no systematic increase in ob-expression in obese children with hypothalamic disease compared to their healthy brothers and sisters. These results show that severe abnormalities involving the ob-gene, analogous to those described in mouse models, are rare in human obesity. We therefore conclude that the cloning and subsequent analysis of the ob-gene has not provided information that can, by itself, explain the genetic component in the development of human obesity.

Randomised placebo-controlled trial of human recombinant insulin-like growth factor I plus intensive insulin therapy in adolescents with insulin-dependent diabetes mellitus.

BACKGROUND: Good glycaemic control in insulin-dependent diabetes mellitus (IDDM) to prevent complications may be difficult to achieve during adolescence, because abnormalities in production of growth hormone or insulin-like growth-factor-I (IGF-I) can lead to lower insulin sensitivity. Recombinant human IGF-I (rhIGF-I) given as an adjunct to insulin therapy in IDDM, might improve glycaemic control in adolescents; we investigated the effects of the addition of IGF-I in a randomised, double-blind, placebo-controlled trial. METHODS: 53 patients with IDDM (26 male, 27 female) with a median age of 16.1 years (range 10.8-20.6) and diabetes of more than 2 years' duration were randomly assigned subcutaneous rhIGF-I (20 or 40 microg/kg daily [n=18, n=18, respectively]) or placebo (n=17), both in addition to multiple-injection insulin therapy for 24 weeks. The primary endpoint, glycated haemoglobin (HbA1c) and routine biochemistry were measured every 4 weeks. Retinal photographs and glomerular-filtration rates were assessed at base line and at the end of the study. Data were analysed by intention to treat. FINDINGS: With a dose of 40 microg/kg rhIGF-I daily, we found significant reductions in HbA1c compared with placebo (p=0.03), without changes in body-mass index, rate of hypoglycaemia, insulin dose, or circulating concentrations of IGF-binding proteins 1 and 3. The greatest median change in HbA1c of -0.6% (range -2.8 to -1.5%) was seen with rhIGF-I 40 microg/kg at week 12, but was not sustained at week 24. The greatest reductions in HbA1c at week 24 were seen among patients with the greatest changes in IGF-I concentrations (r=-0442, p=0.002). Retinal photographs, renal function (glomerular filtration rate and urinary albumin excretion), and routine biochemistry showed no adverse events. INTERPRETATION: Our data confirm that rhIGF-I as an adjunct to insulin therapy can improve HbA1c values in adolescents with IDDM without overt toxic effects, but they raise questions about whether these effects can be sustained in cases of poor compliance or reduced bioefficacy.