Methylation haplotypes of the insulin gene promoter in children and adolescents with type 1 diabetes: Can a dimensionality reduction approach predict the disease?

DNA methylation of cytosine-guanine sites (CpGs) is associated with type 1 diabetes (T1D). The sequence of methylated and non-methylated sites in a specific genetic region constitutes its methyl-haplotype. The aim of the present study was to identify insulin gene promoter (IGP) methyl-haplotypes among children and adolescents with T1D and suggest a predictive model for the discrimination of cases and controls according to methyl-haplotypes. A total of 40 individuals (20 T1D) participated. The IGP region from peripheral whole blood DNA of 40 participants (20 T1D) was sequenced using next-generation sequencing, sequences were read using FASTQ files and methylation status was calculated by python-based pipeline for targeted deep bisulfite sequenced amplicons (ampliMethProfiler). Methylation profile at 10 CpG sites proximal to transcription start site of the IGP was recorded and coded as 0 for unmethylation or 1 for methylation. A single read could result in '1111111111' methyl-haplotype (all methylated), '000000000' methyl-haplotype (all unmethylated) or any other combination. Principal component analysis was applied to the generated methyl-haplotypes for dimensionality reduction, and the first three principal components were employed as features with five different classifiers (random forest, decision tree, logistic regression, Naive Bayes, support vector machine). Naive Bayes was the best-performing classifier, with 0.9 accuracy. Predictive models were evaluated using receiver operating characteristics (AUC 0.96). Methyl-haplotypes '1111111111', '1111111011', '1110111111', '1111101111' and '1110101111' were revealed to be the most significantly associated with T1D according to the dimensionality reduction method. Methylation-based biomarkers such as IGP methyl-haplotypes could serve to identify individuals at high risk for T1D.

Insulin gene promoter methylation status in Greek children and adolescents with Type 1 Diabetes.

The insulin (INS) gene is the one of the most important genes involved in the pathogenesis of Type 1 Diabetes (T1D) after the Major Histocompatibility Complex genes. Studies addressing the issue of hyper- or hypo-methylation status of the INS gene promoter have reported inconsistent results. The majority of studies showed hypomethylation; however a few studies have shown hypermethylation at specific cytosine-guanosine (CpG) sites in the promoter region of the INS gene. The aim of the present study was to analyze the methylation status of the promoter region of the INS gene in Greek children and adolescents with T1D. A total of 20 T1D participants (mean diabetes duration of 6.15±4.12 years) and 20 age- and sex-matched controls were enrolled in the present study. DNA was isolated from whole blood samples, modified using sodium bisulfite and analyzed using PCR and electrophoresis. DNA was then pooled with highly reactive supermagnetic beads at similar molar quantities, submitted for library construction and finally sequenced using next-generation sequencing. The methylation profile at 10 CpG sites around the transcription start site (TSS) of the INS promoter was analysed and expressed as the mean ± standard deviation. The overall mean methylation in patients with T1D did not differ compared with the healthy controls. There was a statistically significant difference between the two groups in hypermethylation at position -345 (P=0.02), while a trend (P=0.06) at position -102 was observed. According to the results of the present study, increased methylation in the INS gene promoter at specific CpG sites around the TSS were already present in childhood T1D. These data may possibly serve as a guide towards the identification of a methylation pattern for detection of development of T1D in genetically predisposed children.

DNA methylation analysis within the IL2RA gene promoter in youth with autoimmune thyroid disease.

BACKGROUND: Alpha-subunit of the interleukin-2 receptor (IL2RA) is involved in the regulation of T-cell function and has been related to autoimmune thyroid disease (AITD). Although the exact mechanisms are not fully understood, promoter methylation might account for differences in gene expression. The aim of this study was to investigate whether there are differences in the percentage of DNA methylation within the IL2RA gene promoter in young patients with AITD. MATERIALS AND METHODS: In a cross-sectional design, the presence of DNA methylation in the IL2RA gene promoter was quantified, by real-time PCR and melting curve analysis, in modified genomic DNA isolated from blood samples of a total of 149 children and adolescents with AITD, including patients with Hashimoto thyroiditis (ΗΤ) (n = 60), Graves' disease (GD) (n = 9), concurrent diagnosis of HT and type 1 diabetes (T1DM + HT) (n = 25), and healthy controls (n = 55). RESULTS: The percentage of DNA methylation in the IL2RA gene promoter was significantly decreased in patients with GD (26.0 ± 4.2%) but not in those with HT (36.3 ± 1.4%) in comparison with controls (41.3 ± 1.5%). CONCLUSIONS: The observed DNA hypomethylation in the IL2RA gene promoter in patients with GD might be related to its increased expression, thus contributing to the etiopathogenesis of GD in childhood and adolescence.

OPG/RANK/RANKL signaling axis in patients with type I diabetes: Associations with parathormone and vitamin D.

BACKGROUND: Type 1 diabetes (T1D) has been associated with a higher fracture risk due to alterations in bone structure and metabolism. On the other hand, the important role of the RANKL/OPG/RANK signaling axis in bone physiology is well established. The aim of this study was to evaluate the levels of receptor activator of nuclear factor kappa-B ligand (RANKL), receptor activator of nuclear factor kappa-B (RANK) and plasma osteoprotegerin (OPG) levels, in T1D youngsters and to investigate factors that could influence the OPG/RANK/RANKL signaling axis such as 25-hydroxy vitamin D [25(OH) D], parathormone (PTH) and age. METHODS: Serum RANKL, RANK, 25(OH) D, PTH levels and plasma OPG levels, were measured in 71 youngsters with T1D and 50 healthy controls matched for age and gender. RESULTS: Plasma OPG levels were significantly lower (p = 0.025) in T1D patients compared to controls. Serum RANKL levels were significantly higher (p = 0.037), while no differences were observed in serum RANK levels (p = 0.946) between the two groups. Serum 25(OH) D levels found significantly decreased (p < 0.001) while serum PTH levels were significantly elevated (p < 0.001) in T1D patients than in controls. CONCLUSIONS: Our results demonstrated that OPG and RANKL may be promising biomarkers for T1D patients. However, their circulating levels were associated with several factors including PTH, 25(OH) D and therefore, may represent an integrative biomarker for a variety of endocrine signaling disturbances observed in T1D.

Testicular microlithiasis in a boy with X-linked adrenal hypoplasia congenita.

X-linked adrenal hypoplasia congenita (AHC) is a rare disorder that usually presents clinically as adrenal insufficiency in early infancy. It is caused by mutations in the NR0B1 gene which is located on the short arm of chromosome X (Xp21). The NR0B1 gene plays an important role in normal development and function of both the adrenal and gonadal axes and some patients with the disease can present in adolescence with hypogonadotropic hypogonadism. Testicular microlithiasis is an ultrasonographic finding of unknown etiology that has been associated with several benign conditions such as cryptorchidism, congenital adrenal hyperplasia, varicoceles, and testicular malignancy. We report the case of an 11-year-old boy who was diagnosed at the age of 8 months with X-linked AHC due to adrenal failure and presented testicular microlithiasis during follow-up. To the best of our knowledge, this is the first case of an X-linked AHC patient diagnosed with testicular microlithiasis in follow-up.

The Intervention of Parents in Supporting of Diabetes Type 1 in Adolescents.

INTRODUCTION: The presence of parents is very important as it enhances the psychology of adolescents resulting to the normal course of the disease. AIM: Investigate the perceptions of adolescents in order to support families in the management of T1D. MATERIAL & METHODS: It is a descriptive study and 56 patients participated. The Diabetes Social Support Questionnaire-Family Version (DSSQ-Family) and some demographic and clinical characteristics were used. The study conducted in the outpatient unit for pediatric diabetology of the 4th Department of Pediatrics in one tertiary General Hospital in a major city of Northern Greece. RESULTS: Younger adolescents felt more supported by their families. Overweight adolescents experienced less support with respect to insulin injections (p = -.333, r = .018), as did the taller respondents (p = -.323, r = .022). Respondents taking more insulin units felt less supported in general (p = -.268, r = .047) and with respect to blood tests (p = -.290, r = .034). Adolescents carrying out more blood glucose measurements felt less supported concerning their meal plan (p =-.307, r = .028), which they rarely complied with (p =-.322, r = .023). CONCLUSIONS: The parental presence is very important enhancing a positive mindset on the part of adolescents and helps achieve the desired treatment results.

46,XY Disorder of Sex Development due to 17-Beta Hydroxysteroid Dehydrogenase Type 3 Deficiency in an Infant of Greek Origin.

17-beta hydroxysteroid dehydrogenase type 3 (17ßHSD-3) enzyme catalyzes the conversion of androstenedione (Δ4) to testosterone (T) in the testes of the developing fetus, thus playing a crucial role in the differentiation of the gonads and in establishing the male sex phenotype. Any mutation in the encoding gene (HSD17B3) can lead to varying degrees of undervirilization of the affected male, ranging from completely undervirilized external female genitalia to predominantly male with micropenis and hypospadias. We present here an infant who was referred to our clinic because of ambiguous genitalia at birth. Gonads were palpable in the inguinal canal bilaterally and no Müllerian structures were identified on pelvic ultrasound. Because of a low T/Δ4 ratio after a human chorionic gonadotropin stimulation test, a tentative diagnosis of 17ßHSD-3 deficiency was made which was confirmed after genetic analysis of the HSD17B3 gene of the patient. The molecular analysis identified compound heterozygosity of two previously described mutations and could offer some further validation for the idea of a founder effect for 655-1;G→A mutation in the Greek population.

Psychological Aspects of Androgen Insensitivity Syndrome: Two Cases Illustrating Therapeutical Challenges.

Androgen Insensitivity Syndrome (AIS) and its heterogeneous phenotypes comprise the pieces of a challenging clinical problem. The lack of standardized guidelines results in controversies regarding the proper diagnostic and therapeutic approach, including the time and type of intervention. Due to its variable phenotype, AIS is not diagnosed at the proper age that would allow optimal psychological and medical support to the patient. Therapeutic approaches are not established, mainly due to the rarity of the disease. In addition, various social and ethical consequences may emerge. The aim of this double case report is to outline the difficulties that may rise during diagnostic, therapeutic, and psychological approach of AIS, especially concerning the handling of the relatives' reaction.

Secondary nocturnal enuresis related to central diabetes insipidus as an early manifestation of intracranial germinomatous germ cell tumors in a series of male youngsters.

Nocturnal enuresis is a common symptom in children. It is usually attributed to benign causes and diagnostic evaluation is not carried out. We report three male young patients initially presenting with short stature and nocturnal enuresis, related to diabetes insipidus, caused by intracranial germinomatous germ cell tumors. In all three cases, water deprivation tests confirmed diabetes insipidus. Extensive endocrinological investigation also showed further hormone deficiencies. Magnetic resonance imaging of the brain revealed the presence of a central nervous system lesion and histology confirmed the final diagnosis. Surgery, radiation with or without chemotherapy was conducted and the patients were treated with hormone replacement therapies. The patients after a long follow-up were free of disease. We present these cases to alert clinicians to bear in mind that the presence of an intracranial germinomatous germ cell tumor should at least be considered in a child presenting with bed wetting, especially if additional symptoms and signs, including late onset puberty and growth delay or morning hypernatremia, may coexist.

Suboptimal glycaemic control enhances the risk of impaired prothrombotic state in youths with type 1 diabetes mellitus.

OBJECTIVE: To estimate markers of prothrombotic state and endothelial dysfunction in youths with type 1 diabetes mellitus (T1DM) and investigate possible associations with anthropometric/demographic data, glycaemic control and lipid profile. METHODS: In a cross-sectional design, we recruited 155 youths with T1DM and determined levels of plasminogen activator inhibitor-1-antigen (PAI-1-Ag), von Willebrand factor-antigen (vWF-Ag), fibrinogen (FB), lipids and glycosylated haemoglobin (HbA1c). RESULTS: Of all the participants, 76 (49%) had increased levels of at least one of prothrombotic factors. Suboptimal glycaemic control was associated with a worse lipid profile and an eightfold increased risk of elevated vWF-Ag levels. Higher vWF-Ag concentrations were also correlated with impaired lipid profile and increased HbA1c values, whereas PAI-1-Ag was positively correlated only with triglyceride levels. After adjustment for potential confounders, only HbA1c contributed independently to the variation in vWF-Ag levels. CONCLUSION: Impaired prothrombotic state and consequently endothelial dysfunction are present in youths with T1DM, representing a cumulative risk factor for future cardiovascular disease (CVD). Achievement and maintenance of euglycaemia and normolipidaemia are crucial to decelerate progress of this process.

Association between magnesium concentration and HbA1c in children and adolescents with type 1 diabetes mellitus.

BACKGROUND: Magnesium levels may be decreased in patients with type 1 diabetes mellitus (T1DM), influencing disease control. Relevant studies concern mainly adults and there are few data from the pediatric population. The aim of the present study was to evaluate magnesium levels and examine their possible association with glycemic control in youths with T1DM. METHODS: In all, 138 children and adolescents with T1DM aged between 1.9 and 20.3 years were recruited to the study. Using a cross-sectional design, we measured anthropometric parameters, HbA1c, serum magnesium, ionized and total calcium, phosphorus, potassium, sodium, and urinary albumin (UA). Estimated glomerular filtration rate (eGFR), based on serum creatinine concentrations, was also calculated. RESULTS: Lower levels of magnesium were found in subjects with poor versus good glycemic control (0.79 ± 0.09 vs 0.82 ± 0.09 mmol/L, respectively; P = 0.002). Serum magnesium levels were negatively correlated with HbA1c (P < 0.001) and positively correlated with UA, calcium, phosphorus, and potassium levels (P < 0.05). After adjustment for confounding factors, only magnesium levels remained significantly associated with HbA1c (adjusted r(2) = 0.172; P = 0.004). The odds ratio for poor glycemic control, indicated by HbA1c >7.5%, between the highest and lowest magnesium concentration quartiles was 0.190 and amounted to a decrease of 1.7% in the HbA1c level. CONCLUSIONS: The present study shows that low serum magnesium levels in children and adolescents with T1DM are associated with an increased risk of poor glycemic control, potentially contributing to the early development of cardiovascular complications.

Development of an electronic web-based software for the management of colorectal cancer target referral patients.

In response to concern about lengthy waiting times for cancer treatment in the UK, the Department of Health introduced 'the colorectal cancer target referral scheme' to improve the referral process for suspected cancer. A user-centred web-based intranet software was developed reflecting the core work of the multi-disciplinary cancer team and the patient journey. The method used was primarily based on the concept of involving the end users (clinicians, nurses, administration staff) in the process of problem definition, software design, formative evaluation, development and implementation, from the very beginning, to ensure its relevance, functionality, and effectiveness. This software improved the interdisciplinary communication among doctors. All patients met the government waiting targets and proved to be a facilitative tool for audit, research and further prospective assessment of our service. Implementing a functional software design is mandatory for the management of target referral patients.