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Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband’s and parent’s(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

To assess the upper body segment (US), lower body segment (LS) and US:LS ratio for children from birth to 18 y of age and to compare these data with other US:LS ratio norms from previously reported studies. A cross-sectional, observational study was conducted in children [birth to 18 y; n = 5454 children (3158 boys)] residing in Pune city, Western India, from December 2015 through June 2017, to evaluate the US, LS and the US:LS ratio. The mean US:LS ratio in boys at birth was 1.45; at one year of age it was 1.35 that decreased to 0.89 at 14 y. The nadir for the US:LS ratio in boys was reached at 14 y of age (0.89). In girls, the mean US:LS ratio at birth was 1.44, at one year of age it was 1.35 that decreased to 0.89 at 12 y. The nadir for the US:LS ratio in girls was reached at 12 y of age (0.89). From 12 to 18 y the US:LS ratio was fairly constant (for girls: 0.89, for boys: 0.9). On plotting the US and LS against the age in present study population, the US and LS curves crossed each other (US:LS = 1) at 7 to 8 y in boys and at 6 to 7 y in girls. The authors describe data for upper and lower body segments and their ratio in boys and girls from birth to 18 y in children from western Maharashtra. These data may help in the diagnosis of disproportionate short stature in children.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Vitamin D, a secosteroid, plays an important role in several physiological processes, and its deficiency can lead to numerous pathophysiological conditions in humans. The primary objective of this study was to develop and validate the robustness of a mass spectrometry-based method capable of quantifying 25(OH)D3 for an upcoming epidemiological survey in India and to pilot test it on healthy volunteers. We first describe the development and validation of various experimental parameters that ascertain the robustness and reliability of 25-hydroxy-vitamin D3 (25(OH)D3) extractions and quantitative measurements from Dried Blood Spot (DBS) samples, where we used eight disks of 3 mm each, punched from the circular spot covering the entire circumference of the spot. Next, we conducted a pilot study, comparing 25(OH)D3 levels from serum and DBS samples from 45 participants using a protocol developed for specifically this purpose. We found that the mean 25(OH)D3 concentrations in DBS samples were comparable to the serum levels (P > 0.05). In summary, our extraction and LC–MS protocol for quantitative 25(OH)D3 measurements are robust and reproducible, and will serve as an invaluable tool for upcoming epidemiological surveys in India and perhaps around the world.

To screen for variants in the MC4R and LEP genes in 46 patients with clinical suspicion of non-syndromic early onset severe obesity (NEOSO). Children with early onset obesity satisfying WHO criteria of obesity were studied. The MC4R and LEP genes were sequenced using a PCR amplicon based NGS on Illumina MiSeq next generation sequencer using an in-house developed protocol. Of the 46 children tested, four were found to have novel pathogenic/likely-pathogenic variants (one in the MC4R gene and three in the LEP gene). In three out of the 4 families, the presence of the variants was confirmed using standard bidirectional capillary sequencing in the probands. Four children with novel likely pathogenic variants in the MC4R and LEP genes are reported. Genetic analysis is crucial in children with early onset obesity and should be considered.

Background: Palm oil contains a high amount of saturated fat compared with most other vegetable oils, but studies have reported inconsistent effects of palm oil on blood lipids. Objective: We aimed to systematically review the effect of palm oil consumption on blood lipids compared with other cooking oils using data from clinical trials. Methods: We searched PubMed and the Cochrane Library for trials of at least 2 wk duration that compared the effects of palm oil consumption with any of the predefined comparison oils: vegetable oils low in saturated fat, trans fat‐containing partially hydrogenated vegetable oils, and animal fats. Data were pooled by using random-effects meta-analysis. Results: Palm oil significantly increased LDL cholesterol by 0.24 mmol/L (95% CI: 0.13, 0.35 mmol/L; I 2 = 83.2%) compared with vegetable oils low in saturated fat. This effect was observed in randomized trials (0.31 mmol/L; 95% CI: 0.20, 0.42 mmol/L) but not in nonrandomized trials (0.03 mmol/L; 95% CI: 20.15, 0.20 mmol/L; P-difference = 0.02). Among randomized trials, only modest heterogeneity in study results remained after considering the test oil dose and the comparison oil type (I 2 = 27.5%). Palm oil increased HDL cholesterol by 0.02 mmol/L (95% CI: 0.01, 0.04 mmol/L; I 2 = 49.8%) compared with vegetable oils low in saturated fat and by 0.09 mmol/L (95% CI: 0.06, 0.11 mmol/L; I 2 = 47.8%) compared with trans fat‐containing oils. Conclusions: Palm oil consumption results in higher LDL cholesterol than do vegetable oils low in saturated fat and higher HDL cholesterol than do trans fat‐containing oils in humans. The effects of palm oil on blood lipids are as expected on the basis of its high saturated fat content, which supports the reduction in palm oil use by replacement with vegetable oils low in saturated and trans fat. This systematic review was registered with the PROSPERO registry at http://www.crd.york. ac.uk/PROSPERO/display_record.asp?ID=CRD42012002601#.VU3wvSGeDRZ as CRD42012002601. JN utr doi: 10.3945/jn.115.210575.