Kah Yin Loke

Procedure for Neonatal Screening for Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

The value of screening of neonates for congenital adrenal hyperplasia is not universally accepted. Procedures for screening are recommended here in order to provide a structure to the testing and ultimately bring together data that will allow the effect of screening to be judged for benefit or dismissed as no better than clinical recognition of the disease state.

Molecular Analysis of CYP-21 Mutations for Congenital Adrenal Hyperplasia in Singapore

Background: Congenital adrenal hyperplasia arising from 21-hydroxylase deficiency is associated with mutations in the CYP21 gene on chromosome 6p. This is the first report on the mutational spectrum of the CYP21 gene in Singapore. Methods: To catalogue the mutations, ten exons of the CYP21 gene from 28 Singaporean patients were analyzed by PCR amplification and direct sequencing. Results: Common mutations in descending order were the intron 2 splice site mutation (32.7% of the alleles), the I172N mutation (23.1% of the alleles), and the R356W mutation (19.2% of the alleles). Two potentially novel mutations were discovered: (1) duplication of 111 bp from codon 21 to codon 57 (exon 1) and (2) missense mutation (L261P, exon 7). There was generally a good genotype-phenotype correlation, allowing accurate prediction of the disease severity.

Association of raised liver transaminases with physical inactivity, increased waist-hip ratio, and other metabolic morbidities in severely obese children.

Objective: To identify factors associated with raised alanine transaminase, aspartate transaminase, and γ-glutaryl transferase in severely obese children Patients and Methods: In all, 201 children with early-onset obesity and greater than 140% ideal weight for height were recruited. Anthropometric and body fat measurements, fasting blood tests, and oral glucose tolerance tests were performed. Results: The mean and standard deviation (SD) for age was 11.1 (3.0) years, for weight for height 170.5% (22.7%), and for percentage body fat was 40.7% (5.2%). Elevated liver transaminases were present in 53 subjects (26.4%), who were therefore at risk for nonalcoholic fatty liver disease, and was associated with male sex (odds ratio [OR] 2.144, 95% confidence interval [CI] 1.033–4.448), Chinese ethnicity (OR 2.062, 95% CI 1.038–4.096), reduced physical activity (OR 2.389, 95% CI 1.163–4.909), insulin resistance (P < 0.05), elevated triglyceride levels (P = 0.029), and increased waist–hip ratio (P = 0.005). Stepwise logistic regression analysis of the main factors as covariates revealed Chinese ethnicity, waist–hip ratio, reduced physical activity, and homeostasis model assessment index were significant predictors. Alanine transaminase/aspartate transaminase were not well correlated with percentage body fat and weight for height. Subjects with type 2 diabetes mellitus and impaired glucose tolerance were more likely to have raised hepatic transaminases (OR 6.176, 95% CI 1.326–28.754). The severity of metabolic syndrome correlated with increasing aspartate transaminase, alanine transaminase, and γ-glutaryl transferase (P < 0.01). Conclusions: Insulin resistance, truncal adiposity, and physical inactivity are major determinants potentially modifiable to reduce risk of nonalcoholic fatty liver disease. Increasing physical activity levels were associated with decreasing insulin resistance and transaminases, despite lack of correlation with waist–hip ratio, which supports the direct benefit of regular physical activity in preventing nonalcoholic fatty liver disease.

Visfatin and its genetic variants are associated with obesity‐related morbidities and cardiometabolic risk in severely obese children

Visfatin is an adipokine, associated with obesity and possibly glucose regulation.

Efficacy and safety of one year of growth hormone therapy in steroid-dependent nephrotic syndrome

OBJECTIVES To study the efficacy and safety of 1 year of growth hormone (GH) therapy in children with steroid-dependent nephrotic syndrome. STUDY DESIGN A prospective pilot, open study in which GH (mean dose 0.32 mg/kg per week) was administered for 1 year to 8 children with steroid-dependent nephrotic syndrome requiring prednisolone (mean dose 0.46 mg/kg per day) to maintain remission. Steroid dependence was defined as recurrence of proteinuria within 2 weeks of discontinuation of prednisolone, or when the dose was lowered below a critical level. At entry, all patients had been steroid dependent for at least 1 year. Anthropometric and bone mineral density measurements after treatment were compared with 1-year pretreatment data. RESULTS Pretreatment mean (+/-SD) chronologic age was 12.6 (+/-3.1) years, with a mean bone age of 9.1 (+/-2.0) years, with delayed puberty in five patients. The mean height velocity increased from 3.7 (+/-1.4) to 9.4 (+/-2.1) cm/yr after 1 year of treatment (p < 0.05). The mean height standard deviation score increased from -1.4 (+/-1.6) to -0.3 (+/-1.1), (p < 0.05). In the spine, the mean bone mineral density increased from 0.50 to 0.64 gm/cm2 (p < 0.05), and in the femoral neck, from 0.55 to 0.64 gm/cm2 (p < 0.05) after 1 year of treatment. Mean lean body mass increased from 58.1% to 62.6% (p < 0.01). There were no significant changes in creatinine clearance, fasting glucose, fasting insulin, or glycosylated hemoglobin levels. The mean bone age increased to 11.4 (+/-2.4) years, and pubertal stage advanced in 2 patients. CONCLUSIONS One year of GH therapy is effective in improving the height standard deviation score, height velocity, bone mineral density, and lean body mass of children with steroid-dependent nephrotic syndrome. There were no significant adverse effects. However, the bone age accelerated at a greater pace than the height age, and further studies are required to define the role of GH therapy in steroid-dependent nephrotic syndrome.

The association of a nucleobindin 2 gene (NUCB2) variant with childhood adiposity.

Nucleobindin 2 (NUCB2) is a precursor of nesfatin-1, a hypothalamic anorectic neuropeptide. The association between variants of the NUCB2 gene and adiposity was examined. 142 severely obese Chinese children in Singapore, and 384 normal weight Chinese children from a longitudinal cohort from Da Qing, China, were studied. NUCB2 was screened using PCR and direct sequencing in 29 severely obese children and 24 non-obese children, then screened for a variant c.1012C>G (Q338E, or rs757081) in the rest of the cohort using TaqMan probe. Five variants, including c.1012C>G (Q338E) were found. Genotyping for c.1012C>G found that the GG genotype was significantly less frequent in the obese group; odds ratio for obese subjects carrying the CC and CG genotypes was 2.29 (95% CI 1.17-4.49) in the dominant model, CC genotype 2.86 (95% CI 1.41-5.81) in the additive model, and C allele 1.57 (95% CI 1.17-2.1). The findings were replicated in an independent cohort of 372 obese and 390 normal weight Chinese children, where the odds ratio of obese subjects with CC and CG genotypes was 1.69 (95% CI 1.12-2.55). Within the Da Qing cohort, subjects with the GG genotype had significantly lower BMI and percentage ideal weight for height (WFH) at 5 and 8years of age. Subjects with lower birth weights also had more pronounced difference in WFH and BMI at 5 and 10years of age between GG subjects versus CC/CG subjects. We postulate that GG genotype is protective against excessive weight gain, and factors which predispose to excessive weight gain such as higher birth weights may ameliorate the effect.

Functional Characterization of Variants in MC4R Gene Promoter Region Found in Obese Children

CONTEXT Mutations in the MC4R gene are the most common cause of monogenic obesity, and there are few studies on mutations in the promoter region. OBJECTIVE The objective of the study was to sequence the promoter region of the MC4R gene in a cohort of obese children to identify rare variants. DESIGN, SETTING, AND PATIENTS A region 1500 bp upstream of the MC4R gene was sequenced in 267 unrelated local children younger than 10 years, with body weight of at least 150% of ideal. An 891-bp upstream region of the MC4R gene was cloned into a luciferase reporter vector for reporter gene assays. INTERVENTIONS There were no interventions. MAIN OUTCOME MEASURES The basal transcriptional activity of the MC4R promoter was analyzed in human embryonic kidney 293 cells using reporter gene assays. RESULTS Three rare variants were detected: c.-803A>G, c.-105C>G, and c.-216C>T. The novel c.-803A>G variant was found in a 9-year-old severely obese Malay boy. This variant was not found in his severely obese mother but was present in his overweight father, who had type 2 diabetes, and also in his normal-weight brother. The novel c.-105C>G variant was found in an obese 9-year-old Malay boy. The c.-216C>T variant was found in an obese Chinese girl with Downs syndrome. The transcriptional activities of the c.-803A>G and c.-105C>G promoters were significantly reduced compared with the wild type but not the c.-216C>T promoter. CONCLUSIONS We have described, for the first time, two novel human MC4R gene promoter variants found in obese children that resulted in a decrease in basal transcriptional activity.

Exogenous Cushing Syndrome: A Lesson of Diaper Rash Cream

A prolonged use of topical corticosteroids can result in Cushing syndrome, though this is less common than with oral or parenteral steroids. Most pediatric cases were due to application of topical steroids for diaper dermatitis. Adverse cardiovascular effects can occur in Cushing syndrome with significant long-term morbidity and mortality, though so far there have been no reports of cardiovascular complications due to excessive usage of topical steroids. We report a 2.5-month-old boy who rapidly developed severe Cushing syndrome induced by the misuse of topical clobetasol, a very potent steroid, without a doctors prescription as a diaper rash cream, and developed moderate left ventricular hypertrophy and pericardial effusion.

Genetic Testing Confirmed the Early Diagnosis of X-Linked Hypophosphatemic Rickets in a 7-Month-Old Infant

Loss-of-function mutations in the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) have been causally associated with X-linked hypophosphatemic rickets (XLHR). The early diagnosis of XLHR in infants is challenging when it is based solely on clinical features and biochemical findings. We report a 7-month-old boy with a family history of hypophosphatemic rickets., who demonstrated early clinical evidence of rickets, although serial biochemical findings could not definitively confirm rickets. A sequencing assay targeting the PHEX gene was first performed on the mother’s DNA to screen for mutations in the 5′UTR, 22 coding exons, and the exon-intron junctions. Targeted mutation analysis and mRNA studies were subsequently performed on the boys’ DNA to investigate the pathogenicity of the identified mutation. Genetic screening of the PHEX gene revealed a novel mutation, c.1080-2A>C, at the splice acceptor site in intron 9. The detection of an aberrant mRNA transcript with skipped (loss of) exon 10 establishes its pathogenicity and confirms the diagnosis of XLHR in this infant. Genetic testing of the PHEX gene resulted in early diagnosis of XLHR, thus enabling initiation of therapy and prevention of progressive rachitic changes in the infant.

A diagnostic curiosity of isolated androstenedione elevation due to autoantibodies against horseradish peroxidase label of the immunoassay

Two sisters with hirsutism presented with mild hirsutism and isolated, grossly elevated (>34.9nmol/L) serum concentrations of androstenedione measured by competitive, homogeneous immunoassay. The clinically discordant laboratory results prompted us to look for assay interference. In this immunoassay, horseradish peroxidase (HRP)-conjugated androstenedione competes with endogenous androstenedione for binding with the solid-phase polyclonal rabbit IgG antibodies. After a wash step, the amount of signal generated by the bound HRP conjugate is inversely proportional to the androstenedione concentration. Alternative analysis by tandem mass spectrometry (a good first line option for troubleshooting) and repeating the competitive immunoassay after polyethylene glycol treatment returned androstenedione concentrations within reference limits. These findings suggested that the original result was spuriously elevated due to assay interference. Additionally, the patient samples were pre-incubated with heterophile blocking reagents, normal rabbit IgG antibodies and HRP-conjugated normal goat IgG antibodies, followed by repeat measurement using the immunoassay. Only samples pre-incubated with HRP-conjugate returned significantly lower androstenedione (9.5 and 12.5nmol/L, respectively), implying neutralisation of the interfering antibodies. Androstenedione remained grossly elevated in the other experiments. This deductive exercise showed that the interference is due to autoantibodies against the HRP label used in the immunoassay. Another immunoassay using HRP label (5α-dihydrotestosterone) also produced gross elevation that was normal by tandem mass spectrometry analysis. Assay interferences, though not uncommon, are frequently overlooked. Laboratory results discordant with clinical features should prompt consideration of assay interference to avoid unnecessary investigations and treatment. This is the first report of autoantibodies against the HRP label used in immunoassay.