High Sclerostin and Dickkopf-1 (DKK-1) Serum Levels in Children and Adolescents With Type 1 Diabetes Mellitus.

Context: Childhood type 1 diabetes mellitus (T1DM) is associated with decreased bone mass. Sclerostin and dickkopf-1 (DKK-1) are Wnt inhibitors that regulate bone formation. Objective: To evaluate sclerostin and DKK-1 levels in T1DM children and to analyze the influence of glycemic control on bone health. Design and setting: Cross-sectional study conducted at a clinical research center. Participants: One hundred and six T1DM subjects (12.2 ± 4 years), 66 on multiple daily injections (MDIs) and 40 on continuous subcutaneous infusion of insulin (CSII), and 80 controls. Results: The average bone transmission time (BTT) and amplitude-dependent speed of sound (AD-SoS) z scores were lower in patients with diabetes than in controls. Significantly increased DKK-1 (3593 ± 1172 vs 2652 ± 689 pg/mL; P < 0.006) and sclerostin (29.45 ± 12.32 vs 22.53 ± 8.29; P < 0.001) levels were found in patients with diabetes with respect to controls, particularly in patients on MDI compared with ones on CSII. Glycemic control was improved in CSII patients compared with MDI ones (P < 0.001) and was also associated with significantly higher BMI-SDS (P < 0.002) and BTT z scores (P < 0.02). With adjustment for age, multiple linear regression analysis of DKK-1 and sclerostin as dependent variables showed that levels of glycated hemoglobin, glucose, 25(OH) vitamin D, osteocalcin, and parathyroid hormone; years of diabetes; and BMI-SDS and AD-SoS z score were the most important predictors (P < 0.0001). Conclusions: Our study highlighted (1) the high serum levels of DKK-1 and sclerostin in T1DM children and their relationship with altered glycemic control and (2) the effect of CSII on improvement of glycemic control and bone health in T1DM children.

Bone Fragility in Turner Syndrome: Mechanisms and Prevention Strategies.

Bone fragility is recognized as one of the major comorbidities in Turner syndrome (TS). The mechanisms underlying bone impairment in affected patients are not clearly elucidated, but estrogen deficiency and X-chromosomal abnormalities represent important factors. Moreover, although many girls with TS undergo recombinant growth hormone therapy to treat short stature, the efficacy of this treatment on bone mineral density is controversial. The present review will focus on bone fragility in subjects with TS, providing an overview on the pathogenic mechanisms and some prevention strategies.

Mechanisms of enhanced osteoclastogenesis in girls and young women with Turner's Syndrome.

Subjects with hypergonadotropic hypogonadism due to Turner's syndrome show low cortical mineral density, osteoporosis and risk of fractures. It is not clear if this bone fragility derives from chromosomal abnormalities or is the result of inadequate bone formation due to estrogen deficiency. The aim of this study was to investigate the cellular mechanisms underlying bone fragility in subjects with Turner's syndrome before induction of puberty and after hormonal replacement therapy (HRT). For this purpose, we have evaluated the osteoclastogenic potential of non-fractioned and T-cell depleted cultures of peripheral blood mononuclear cells (PBMCs) belonging to girls with Turner's syndrome who had not been treated with HRT yet, girls and young women who were on HRT and age-matched controls. Untreated subjects showed high FSH serum levels, whereas the other subjects displayed normal FSH serum levels. T-cell immunophenotype was analyzed through flow cytometry. Biochemical and DXA analyses were performed. Spontaneous osteoclastogenesis in non-fractioned and T-cell depleted cultures of PBMC belonging to girls with high FSH levels was more evident than in cultures of subjects with normal FSH levels. In the former, osteoclastogenesis was sustained by monocytes expressing high levels of c-fms, TNF-α and RANK, and T-cells producing high RANKL and TNF-α; in the latter it was supported by T-cells expressing high RANKL levels. CD4(+)CD25(high) T-cells were reduced in all subjects, whereas CD3(+)/CD16(+)/CD56(+) NKT-cells were increased in those with high FSH levels. High RANKL and CTX levels were detected in the sera. Bone impairment was already detectable by DXA in subjects aged under 10, although it became more evident with aging. In conclusion, our results demonstrated that bone fragility in subjects with Turner's syndrome is associated to enhanced osteoclastogenesis. This process seems to be due to high FSH serum levels before HRT, whereas it is caused by high RANKL during HRT.

The p53 family member p73 modulates the proproliferative role of IGFBP3 in short children born small for gestational age.

The regulation of insulin-like growth factor-binding protein 3 (IGFBP3) gene expression is complex, because it can be induced by agents that both stimulate and inhibit the proliferation. The principal aim of this study was to investigate whether p73, a member of the p53 gene family, has a role in the regulation of the IGFBP3 expression and whether this regulation occurs in a context of cell survival or death. We demonstrate that IGFBP3 is a direct TAp73α (the p73 isoform that contains the trans-activation domain) target gene and activates the expression of IGFBP3 in actively proliferating cells. As IGFBP3 plays a key role in regulating the growth hormone/insulin-like growth factor type 1 (GH/IGF1) axis, whose alterations in gene expression appear to have a role in the growth failure of children born small for gestational age (SGA), we measured the mRNA expression levels of p73 and IGFBP3 in a group of SGA children. We found that mRNA expression levels of p73 and IGFBP3 are significantly lower in SGA children compared with controls and, in particular, p73 mRNA expression is significantly lower in SGA children with respect to height. Our results shed light on the intricate GH/IGF pathway, suggesting p73 as a good biomarker of the clinical risk for SGA children to remain short in adulthood.

Skeleton and glucose metabolism: a bone-pancreas loop.

Bone has been considered a structure essential for mobility, calcium homeostasis, and hematopoietic function. Recent advances in bone biology have highlighted the importance of skeleton as an endocrine organ which regulates some metabolic pathways, in particular, insulin signaling and glucose tolerance. This review will point out the role of bone as an endocrine "gland" and, specifically, of bone-specific proteins, as the osteocalcin (Ocn), and proteins involved in bone remodeling, as osteoprotegerin, in the regulation of insulin function and glucose metabolism.

Metabolic syndrome in childhood leukemia survivors: a meta-analysis.

A significant number of long-term complications have been described in childhood leukemia survivors. In particular, these patients may present features of metabolic syndrome (MetS), and therefore increased risk for cardiovascular diseases. The aim of this meta-analysis is to evaluate the prevalence and the risk of MetS in survivors of childhood leukemia. Two authors independently performed a systematic literature search in PubMed and EMBASE to March 2014, reviewed and selected articles, based on pre-determined selection criteria. Twelve articles, comprising 2,337 participants (1,462 cases and 875 controls), were included in the meta-analysis. Only three of them were case-control studies eligible for the meta-analysis. The childhood leukemia survivors showed an increased risk of MetS as compared to healthy controls (OR = 4.36; 95 % CI 1.19-16.22). The risk was significantly increased only in patients treated with chemotherapy and radiotherapy (OR = 7.79; 95 % CI 1.27-47.77), and not in patients treated with only chemotherapy (OR = 2.35; 95 % CI 0.40-13.78). Childhood leukemia survivors, in particular if treated also with radiotherapy, are prone to develop MetS more than healthy controls. Monitoring of MetS components in these patients is necessary to avoid cardiovascular consequences later in life.

Osteoclastogenic potential of peripheral blood mononuclear cells in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterized by hypoplastic or aplastic clavicles, dental abnormalities, and delayed closure of the cranial sutures. In addition, mid-face hypoplasia, short stature, skeletal anomalies and osteoporosis are common. We aimed to evaluate osteoclastogenesis in a child (4 years old), who presented with clinical signs of CCD and who have been diagnosed as affected by deletion of RUNX2, master gene in osteoblast differentiation, but also affecting T cell development and indirectly osteoclastogenesis. The results of this study may help to understand whether in this disease is present an alteration in the bone-resorptive cells, the osteoclasts (OCs). Unfractionated and T cell-depleted Peripheral Blood Mononuclear Cells (PBMCs) from patient were cultured in presence/absence of recombinant human M-CSF and RANKL. At the end of the culture period, OCs only developed following the addition of M-CSF and RANKL. Moreover, real-time PCR experiment showed that freshly isolated T cells expressed the osteoclastogenic cytokines (RANKL and TNFα) at very low level, as in controls. This is in accordance with results arising from flow cytometry experiments demonstrating an high percentage of circulating CD4(+)CD28(+) and CD4(+)CD27(+) T cells, not able to produce osteoclastogenic cytokines. Also RANKL, OPG and CTX serum levels in CCD patient are similar to controls, whereas QUS measurements showed an osteoporotic status (BTT-Z score -3.09) in the patient. In conclusions, our findings suggest that the heterozygous deletion of RUNX2 in this CCD patient did not alter the osteoclastogenic potential of PBMCs in vitro.

Treatment of osteoporosis in children with glucocorticoid-treated diseases.

Glucocorticoid induced osteoporosis (GIO) is the most frequent form of drug induced osteoporosis. Glucocorticoids affect osteoblastogenesis, osteoclastogenesis and promote the apoptosis of osteoblasts and osteocytes. A decrease of bone mineral density has been described in several pediatric diseases that require glucocorticoids, both as long-term replacement therapy, such as Congenital Adrenal Hyperplasia, and as treatment of acute phase or relapses, such as asthma, juvenile rheumatoid arthritis, inflammatory bowel diseases, systemic lupus erythematosus, organ transplantation and Steroid Sensitive Nephrotic Syndrome. The increasing number of children with GIO and at risk of fractures reflects the complex nature of this condition, and the need of development of anti-osteoporotic drugs. In this review, we focus on the mechanisms of GIO in some pediatric diseases and on treatment of osteoporosis. We also report data on new signaling pathways as potential targets for future anti-osteoporotic drugs.

Postmenopausal osteoporosis: the role of immune system cells.

In the last years, new evidences of the relationship between immune system and bone have been accumulated both in animal models and in humans affected by bone disease, such as rheumatoid arthritis, bone metastasis, periodontitis, and osteoporosis. Osteoporosis is characterized by low bone mass and microarchitectural deterioration of bone tissue with a subsequent increase in bone fragility and susceptibility to fractures. The combined effects of estrogen deprivation and raising of FSH production occurring in menopause cause a marked stimulation of bone resorption and a rapid bone loss which is central for the onset of postmenopausal osteoporosis. This review focuses on the role of immune system in postmenopausal osteoporosis and on therapeutic strategies targeting osteoimmunology pathways.

Effect of recombinant insulin-like growth factor-1 treatment on short-term linear growth in a child with Majewski osteodysplastic primordial dwarfism type II and hepatic insufficiency.

We report the case of a boy affected by severe intrauterine and postnatal growth retardation, microcephaly, facial dysmorphisms and postnecrotic cirrhosis, diagnosed at birth as having Seckel syndrome, and subsequently confirmed as Majewski osteodysplastic primordial dwarfism type II (MOPD II) on the basis of clinical and radiological features of skeletal dysplasia. At our observation (6 years 7 months) he presented height -10.3 standard deviation score (SDS), weight -22.1 SDS, head circumference -8 SDS, delayed bone age of 4 years with respect to chronological age. In consideration of the low levels of insulin-like growth factor-1 (IGF-1) as well as of hepatic insufficiency, we started the treatment with recombinant human IGF-1 (rhIGF-1) at the dose of 0.04 mg/kg in 2 doses/day, with an increase of 0.04 mg/kg after 1 week until the maximum dose of 0.12 mg/kg. We observed an early response to rhIGF-1 treatment, with a shift of height velocity from 1.8 cm/year (-4.6 SDS) at 4 cm/year (-1.9 SDS), and an increase in bone age of 1.5 years during the first 6 months. rhIGF-1 treatment does not seem to be able to replace the physiological action of IGF-1 in patients with MOPD II and hepatic insufficiency, however, it seems to preserve the typical growth pattern of MOPD II patients, avoiding a further widening of the growth deficiency in these subjects.

Glucocorticoid-induced osteoporosis in children with 21-hydroxylase deficiency.

21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2). Children with 21-OHD need chronic glucocorticoid (cGC) therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO) is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD.

Nonalcoholic fatty liver disease in prepubertal children born small for gestational age: influence of rapid weight catch-up growth.

BACKGROUND/AIMS: We studied the association of low birth weight with ultrasound-assessed nonalcoholic fatty liver disease (NAFLD) to test the hypothesis that fetal growth retardation followed by a rapid weight catch-up growth might be an additional factor responsible for liver steatosis via insulin resistance (IR) and/or intra-abdominal fat. METHODS: We enrolled 23 children born small for gestational age (SGA) with a rapid catch-up growth within the first 6-12 months, and 24 appropriate for gestational age (AGA) children as controls. All children underwent anthropometric, body composition measurements and evaluation of liver function tests, lipid profile, plasma glucose and insulin levels. Abdominal ultrasonography was performed in order to asses liver steatosis and thickness of subcutaneous and visceral adipose tissue. RESULTS: NAFLD were observed in 8 out of the 23 SGA children (34.8%). IR and visceral fat were significantly increased in children with hepatic steatosis compared to those without. IR index was significantly related to liver steatosis, independently of body mass index standard deviation score and visceral fat. CONCLUSIONS: NAFLD should be recognized as an emerging problem in SGA prepubertal children who presented a rapid weight gain in postnatal life, and IR plays the key role. An appropriate diet during pregnancy and in the first year of life might prevent metabolic syndrome and NAFLD in these subjects.

Risk factors for subclinical atherosclerosis in diabetic and obese children.

BACKGROUND: Increased carotid intima-media thickness (cIMT) is considered a marker of early-onset atherosclerosis and it seems to predict cardiovascular events both in obese and diabetic subjects. We aimed to evaluate early signs of atherosclerosis and investigate for predisposing factors in children and adolescents affected by type 1 diabetes (T1DM) or obesity, comparing them with healthy controls. METHODS: Out of 71 enrolled subjects (mean age 12.8 ± 2.3 years), 26 had T1DM and 24 were obese, while 21 age- and sex-matched subjects acted as controls. cIMT was measured using standardized methods. Serum glucose, insulin, cholesterol, triglycerides and C-reactive protein levels were evaluated. An oral glucose tolerance test (OGTT) was performed in obese subjects. RESULTS: Diabetic and obese individuals showed higher cIMT mean values than healthy controls (p < 0.005). cIMT of the three examined segments correlated positively with fasting glucose levels and negatively with units of insulin/kg/day administered in T1DM individuals. A positive correlation between insulin levels (basal and after oral glucose load) and cIMT of common, internal and external carotid artery was found in obese subjects (p < 0.03). High density cholesterol levels represented a protective factor for cIMT in this latter group of the study population. CONCLUSIONS: Our findings show that cIMT correlates with high insulin levels (a sign of insulin resistance) in obese patients and with high fasting glucose levels (a sign of relative insulin deficiency) in T1DM subjects, confirming the need of reducing hyperinsulinism and monitoring blood glucose levels in these subjects to prevent atherosclerosis.

High dickkopf-1 levels in sera and leukocytes from children with 21-hydroxylase deficiency on chronic glucocorticoid treatment.

Children with 21-hydroxylase deficiency (21-OHD) need chronic glucocorticoid (cGC) therapy to replace congenital deficit of cortisol synthesis, and this therapy is the most frequent and severe form of drug-induced osteoporosis. In this study, we enrolled 18 patients (9 females) and 18 sex- and age-matched controls. We found in 21-OHD patients high serum and leukocyte levels of dickkopf-1 (DKK1), a secreted antagonist of the Wnt/ß-catenin signaling pathway known to be a key regulator of bone mass. In particular, we demonstrated by flow cytometry, confocal microscopy, and real-time PCR that monocytes, T lymphocytes, and neutrophils from patients expressed high levels of DKK1, which may be related to the cGC therapy. In fact, we showed that dexamethasone treatment markedly induced the expression of DKK1 in a dose- and time-dependent manner in leukocytes. The serum from patients containing elevated levels of DKK1 can directly inhibit in vitro osteoblast differentiation and receptor activator of NF-κB ligand (RANKL) expression. We also found a correlation between both DKK1 and RANKL or COOH-terminal telopeptides of type I collagen (CTX) serum levels in 21-OHD patients on cGC treatment. Our data indicated that DKK1, produced by leukocytes, may contribute to the alteration of bone remodeling in 21-OHD patients on cGC treatment.

Oxidative stress in obesity and metabolic syndrome in children and adolescents.

BACKGROUND/AIMS: The aim of this study was to investigate the alterations in the oxidant/antioxidant status in obese children with and without metabolic syndrome (MetS). METHODS: We recruited 25 Caucasian obese children with MetS, 30 Caucasian children with simple obesity and a control group of 30 Caucasian children. We performed diacron-reactive oxygen metabolites (d-ROMs) test and biological antioxidant potential (BAP) test in order to evaluate the oxidant-antioxidant status in recruited patients. RESULTS: d-ROM level was significantly higher in obese children with and without MetS (p = 0.005). The total antioxidant capacity (BAP level) was reduced in MetS and noMetS children compared to controls (p = 0.009). The subjects without MetS had higher d-ROMs test and lower BAP/d-ROMs ratio than subjects with MetS (although not significant). The ratio BAP/d-ROMs was higher in controls than noMetS and MetS children (p < 0.0001). d-ROM level was higher in prepubertal subjects with MetS than pubertal ones (p = 0.03). A direct correlation was found between d-ROM levels and BMI SDS (p = 0.0005), while an inverse correlation was found between BAP and BMI SDS (p = 0.004) and BAP/d-ROMs and BMI SDS (p = 0.0001). CONCLUSIONS: This result confirms that fat accumulation plays a key role in the pathogenesis of systemic oxidative stress already during pediatric age.

Genotype-phenotype correlation in juvenile Paget disease: role of molecular alterations of the TNFRSF11B gene.

Juvenile Paget disease (JPD) {MIM 239000} is a rare inherited bone disease that affects children. The patients affected with JPD present an altered bone turnover, therefore, show a phenotype characterized by progressive bone deformities, fractures, and short stature. Deletions or missense mutations of the TNFRSN11B gene are common in these children. This gene encodes a soluble protein, the osteoprotegerin, which leads to uncontrolled osteoclastogenesis when mutated. JPD is characterized by a strong genotype-phenotype correlation, so depending on the alteration of the TNFRSN11B gene, the phenotype is variable. This review describes the different clinical features which are characteristic of JPD and the correspondence with the different molecular alterations of the TNFRSN11B gene.

Analysis of endothelial protein C receptor gene and metabolic profile in Prader-Willi syndrome and obese subjects.

The endothelial protein C receptor (EPCR) has a critical role in the regulation of anticoagulant and anti-inflammatory functions of activated protein C (APC). Abnormalities in EPCR might be associated with an increased risk of thrombosis. In this respect, a 23 bp insertion in the exon 3 of the EPCR gene predicts a truncated protein which cannot bind APC. High levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are found both in the obese and in subjects with Prader-Willi syndrome (PWS). Several cardiovascular risk factors are already present in prepubertal PWS children, but it is uncertain which mechanism contributes to the increased risk of cardiovascular disease in PWS. We analyzed the distribution of 23 bp insertion in the EPCR gene in 81 overweight and obese PWS subjects, 52 adults and 29 children, and in 58 overweight and obese children and adolescents (controls). We found that 1/58 (1.7%) of the controls was heterozygous for the 23 bp insertion, while this mutation was never found in PWS subjects. Furthermore, we evaluated CRP levels, glucose, insulin, and lipid profile, and we found higher CRP values in PWS adults with respect to children with PWS and controls, and a better insulin sensitivity in all PWS subjects than in the controls. This study suggests that in PWS subjects there is no predisposition to develop thrombotic events in association with EPCR gene alteration and demonstrates substantial differences regarding metabolic and inflammatory profile between PWS and non-PWS obese children, with further impairment in adults with PWS.

IGF2 gene variants and risk of hypertension in obese children and adolescents.

Obese children have a great risk of hypertension and cardiovascular morbidity in adults. The insulin-like growth factor type II (IGF-II) regulates glucose homeostasis, cardiovascular functions, and lipid metabolism. IGF2 gene variants have shown a strong association with weight, body mass index (BMI), and metabolic profile in adults. We performed the molecular screening of two IGF2 polymorphisms (6815 A/T, 820 G/A), in 227 obese children to evaluate the potential association between IGF2 variants with either obesity or high blood pressure (assessed with a 24-h holter system) or both. A second cohort of age-, sex-, and BMI-matched children were enrolled to confirm any eventual association. We observed a significant association between the 6815 A/T IGF2 gene variant and high systolic blood pressure in obese children. Homozygote subjects for the T6815 allele showed, even in 24-h measurements, a higher risk to develop hypertension than those carrying the A6815 allele (OR = 3.7, 95% CI: 1.59-8.66). This result was confirmed in the second cohort (OR = 4.1, 95% CI: 1.41-6.50). Any statistically significant difference in terms of BMI between the genotype groups was observed. Our results suggest that IGF2 gene variants are involved in the blood pressure regulation in obese children.

Molecular characterization of a large cohort of patients with Chronic Granulomatous Disease and identification of novel CYBB mutations: an Italian multicenter study.

Chronic Granulomatous Disease (CGD) is a rare inherited disorder in which phagocytes fail to produce antimicrobial superoxide because NADPH oxidase activity is absent. In about 65% of the cases, the disease is due to mutations affecting the X-linked CYBB gene, encoding the gp91(phox) subunit of NADPH oxidase. We investigated 34 CGD male patients by DHPLC and direct sequencing. A mutation was found in the CYBB gene of 33 patients and 9 of these were novel: one non-sense mutation (c.1123 G>T), three missense mutations (c.58G>A; c.1076 G>C; c.1357 T>A), two splice site mutations (c.141+5G>T; c.142-1G>A), one duplication (c.42_45dupCATT), one deletion (c.184delT), and one rare deletion of two non-contiguous nucleotides (c.1287delT+c.1290delC). One patient had the most frequent GT homozygous deletion in exon2 of the NCF-1 gene encoding the p47(phox) subunit of NADPH oxidase. The carrier analysis was performed in 23 patients' mothers and 16 female relatives through molecular and FISH studies. No clear correlation between the severity of clinical symptoms and the type of mutation could be demonstrated. This study further supports the great heterogeneity of the disease and the notion that genetic analysis is a critical step in obtaining a definitive diagnosis for CGD.