Saika Iwama

Association of HLA-DR, -DQ Genotype and CTLA-4 Gene Polymorphism with Graves’ Disease in Japanese Children

Objective: Childhood onset Graves’ disease (GD) has been documented to be clinically distinct from adult onset GD, and an association with the genes encoding HLA and CTLA-4 (cytotoxic T lymphocyte antigen-4) has been reported in both Caucasian and Japanese adult GD patients. The aim of this study was to determine whether HLA-DR, -DQ and CTLA-4 are associated with childhood onset GD in Japanese individuals. Methods: We investigated the genotype of HLA class II (DRB1, DQB1) and the A/G transition polymorphism of CTLA-4 exon 1 position 49 in 43 GD patients and in healthy controls for comparison. The CTLA-4 alleles were identified by the polymerase chain reaction (PCR) of genomic DNA and restriction fragment-length polymorphism analysis (PCR-RFLP) with Ita1. Results: The frequency of both HLA-DRB1*0405 and DQB1*0401 was increased in the patient group (DRB1*0405: 26.7%, p < 0.001; DQB1*0401: 25.6%, p < 0.005) compared with the controls. Patients with GD had a significantly lower frequency of the AA genotype of CTLA-4 than the controls, but there was no difference in allele frequency between the G and A allele. Conclusions: the association of HLA-DRB1 and DQB1 genotype with susceptibility to childhood onset GD differs from that in adult onset GD, whereas the association between CTLA-4 gene polymorphism and childhood onset GD is similar to that in adult onset GD in Japanese individuals, but the association is weak.

Short-Term Secretory Regulation of Ghrelin during Growth Hormone Provocative Tests in Prepubertal Children with Various Growth Hormone Secretory Capacities

Background and Objective: Ghrelin is a novel gastric peptide which stimulates GH secretion and has been demonstrated to have orexigenic and adipogenic properties. Insulin is a physiological and dynamic modulator of plasma ghrelin, and insulinemia possibly mediates the effect of the nutritional state on the plasma concentrations of ghrelin in adults. No data on the regulation of GH secretion by ghrelin have so far been reported, nor has the possible influence of hypoglycemia on the plasma ghrelin levels in children been reported. Methods: Provocative studies were performed using a variety of stimuli, including insulin-induced hypoglycemia, and glucagon, arginine and L-dopa loading. We studied a group of 27 children with short stature being investigated for GH deficiency (10 F, 17 M; age 4–14 years; height SDS –0.92 to –3.27); the subjects were instructed to fast overnight, and the following morning, the relationships among the plasma ghrelin, GH and glucose levels were investigated by determining the plasma ghrelin profiles during those provocative tests. Using a new method for determining the two types of ghrelin, samples were obtained for determination of the plasma ghrelin, serum glucose and serum GH levels after the administration of the aforementioned stimulating agents. Results: All the four stimuli caused a significant decrease in the circulating C- and N-ghrelin levels with a nadir at +30 min, with the exception of the N-ghrelin level following the L-dopa loading. During the same period, the plasma GH level increased following insulin, arginine and L-dopa loading, and the plasma glucose level increased significantly following glucagon loading. In the arginine and L-dopa load connected, a significant correlation was observed between the 30-min change in the serum GH level and the 30-min change in the plasma C-ghrelin level. In the multiple regression analysis to explain the 30-min change in the plasma level of C-ghrelin, the baseline plasma level of C-ghrelin (basal), height and % overweight were the only three significant parameters, accounting for 85.2% of the variance. Conclusion: This study demonstrated that the inverse relation between the circulating GH and ghrelin levels may indicate the existence of a feedback loop, and also lends support to the assumption of a GH-independent relationship between plasma ghrelin and glucose levels. These observations constitute further evidence to suggest that peripheral ghrelin is a direct growth-promoting hormone.

Genetic factors and clinical significance of acanthosis nigricans in obese Japanese children and adolescents.

Aim: To clarify the clinical significance of acanthosis nigricans (AN) and the association of gene polymorphisms in the β2‐ and β3‐adrenergic receptors (B2ADR and B3ADR) in Japanese obese children and adolescents. Methods: Seventy obese subjects (56 boys, 14 girls) from 5 to 19 y of age were examined as to clinical features. Genetic analyses were performed in 83 obese subjects (61 boys, 22 girls), 2 to 17 y of age. Typing of gene polymorphisms in B2ADR and B3ADR was achieved by polymerase chain reaction (PCR) of genomic DNA and restriction fragment‐length polymorphism analysis (PCR‐RFLP). Results: The group with AN (n=30) had higher values for percent overweight, BMI, waist circumference, fasting insulin, HOMA‐R, leptin and PAI‐1 than the AN‐negative group (n=40), but there were no significant differences in age, sex or percent body fat between the two groups. The prevalences of B2ADR Gly16 and B3ADR Arg64 were significantly higher in AN‐positive (n=26) than in AN‐negative (n=57) subjects. In addition, the AN frequency was significantly higher in the group with both Gly16 and Arg64 than in the group with neither of these alleles (55.6% vs 12.5%, p < 0.05).

Impact of polymorphisms of β2- and β3-adrenergic receptor genes on longitudinal changes in obesity in early childhood

Sir, Few studies have been conducted on the effects of these polymorphisms on obesity in children. Obesity is influenced by multiple behavioural, environmental and genetic factors (1). This study was conducted to ascertain whether polymorphisms of the 2and 3adrenergic receptor (AR) subtypes influence the development of obesity over time in early childhood. This was a descriptive study using the medical charts of 46 children (19 girls and 27 boys) who were of short stature and treated by growth hormone (GH). All the children enrolled in the study were diagnosed as having partial but not complete GH deficiency, and were well nourished. The thyroid, kidney and liver functions were normal, and none of the children had celiac disease. Anthropometric data of the children were recorded longitudinally from 1 to 6 y of age prior to the commencement of GH treatment. The severity of obesity was expressed as the percent overweight, which represents the percentage deviation from the ideal body weight based on age, gender and stature. Standing height and body weight were measured by trained examiners following identical protocols over the entire course of the study. Age-stratified analysis was used to examine longitudinally, over a 5-y period, the effects of genetic polymorphisms on obesity in 46 preschool children of short stature with GH deficiency. 2-AR gene codon16 and 3-AR gene codon64 polymorphisms were analysed by the PCR-RFLP method. The results of the 2and 3-AR genotyping are shown in Table 1. No difference in the frequency distribution of the 2and 3-AR genotyping was observed compared with that of Japanese non-diabetic subjects (2, 3). The mean percent overweight increased linearly during the 5-y study period, from 5.3% to 17.7% in the group of children with the mutation of the AR codon64 polymorphism (Trp/Arg and Arg/Arg, n = 19), and 5.0% to 1.3% in those without this mutation (Trp/Trp, n = 27, because of the limited number of Arg/Arg subjects). These differences attained statistical significance at 4.5 and 6 y of age. In contrast, the differences among the subtypes of the 2AR gene codon16 polymorphism did not attain statistical significance at any time during this study. Trp64Arg polymorphism of the 3-AR gene appears to be a genetic risk factor for obesity in Japanese preschool children, while the Arg16Gly polymorphism of the 2-AR gene does not appear to be associated with childhood obesity.

Absence of Heterozygous K83E and R257X Mutations of the AIRE-1 Gene in 46 Children with Type 1 Diabetes and 44 Children with Graves' Disease

Type 1 diabetes mellitus (DM) and Graves’ disease are autoimmune diseases, and a number of genetic factors, including HLA and CTLA-4 genes, have been reported to contribute to their etiology. The gene responsible for autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy (APECED) has been cloned and named the autoimmune regulator-1 (AIRE-1) gene. AIRE-1 protein is thought to be a transcription regulatory protein and to have a role in the maintenance of immunological tolerance. The aim of this study was to determine whether heterozygous AIRE-1 gene mutations are associated with childhood-onset type 1 diabetes and Graves’ disease in the Japanese population. We investigated 46 children with type 1 DM (29 females and 17 males; age at the time of diagnosis, 0.5–16 yr) and 44 children with Graves’ disease (34 females and 10 males; age at the time of diagnosis, 3–16 yr) for the presence of the K83E mutation in exon 2 and the R257X mutation in exon 6 of the AIRE-1 gene. The alleles were identified by polymerase chain reaction of genomic DNA and restriction fragment-length polymorphism analysis (PCR-RFLP) with endonuclease TaqI. Since no patients with type 1 DM or Graves’ disease were found to carry the K83E or the R257X heterozygous mutation, we concluded that neither the K83E nor the R257X heterozygous mutation in the AIRE-1 gene seem to be the cause of the more common isolated endocrinopathies, i.e., type 1 diabetes mellitus and Graves’ disease, in Japanese children.