Neonatal Screening for Congenital Adrenal Hyperplasia in Indian Newborns with Reflex Genetic Analysis of 21-Hydroxylase Deficiency.

Congenital adrenal hyperplasia (CAH), screened for in neonates, is the second most common endocrinopathy after congenital hypothyroidism.Newborn screening for CAH due to CYP21A2 deficiency is performed by immunologic assay for 17-hydroxyprogesterone (17-OHP). The second-tier test for confirmation of diagnosis is carried out on recall venous blood sample from screen positives measuring 17-OHP, or other metabolites of steroid metabolism by liquid chromatography-tandem mass spectroscopy. However, as steroid metabolism is dynamic, it can affect these parameters even in the recall sample of a stressed neonate. Moreover, there is some time delay in recalling the neonate for repeat testing. Reflex genetic analysis of blood spot from the initial Guthrie cards of screen positive neonates, if used for confirmatory testing, can avoid this time delay as well as the effect of stress on steroid metabolism. In this study, we used Sanger sequencing and MLPA in a reflex manner for molecular genetic analysis to confirm CYP21A2-mediated CAH. Out of 220,000 newborns screened, 97 were positive on the initial biochemical screen, of which 54 were confirmed true positives with genetic reflex testing, giving incidence of CAH as 1:4074. Point mutations were more common than deletions, indicating that Sanger sequencing should be used ahead of MLPA for molecular diagnosis in India. Amongst the variants detected, the most common was I2G-Splice variant (44.5%), followed by c.955C>T (p.Gln319Ter) (21.2%); Del 8 bp and c.-113G>A were detected with frequencies of 20.3% and 20%, respectively. In conclusion, reflex genetic testing is an effective strategy for identifying true positives in CAH screening in neonates. This will obviate need for recall samples and also aid effective counselling and timely prenatal diagnosis in the future. In Indian newborns, as point mutations are more common than large deletions, Sanger sequencing should be the initial method of choice for genotyping, ahead of MLPA.

Gas chromatography/mass spectrometry-based urine metabolome study in children for inborn errors of metabolism: An Indian experience.

OBJECTIVE: The present study highlights the feasibility of gas chromatography/mass spectrometry (GC/MS)-based analysis for simultaneous detection of >200 marker metabolites in urine found in characteristic pattern in inborn errors of metabolism (IEM) in India. DESIGN AND METHODS: During this retrospective study conducted from July 2013 to January 2016, we collected urine specimens on filter papers from Indian children across the country along with relevant demographic and clinical data. The laboratory technique involved urease pretreatment followed by deproteinization, derivatization, and subsequent computer-aided analysis of organic acids, amino acids, fatty acids, and sugars by GC/MS, which enable chemical diagnosis of IEM. RESULTS: Totally 23,140 patients were investigated for IEM with an estimated frequency of about 1.40%, that is, 323 positive cases. Most frequent disorders observed were of primary lactic acidemia (27.2%) and organic acidemia (methylmalonic aciduria, glutaric acidemia type I, propionic aciduria, etc.) followed by aminoacidopathies (maple syrup urine disease, phenylketonuria, tyrosinemia, etc.). Furthermore, alkaptonuria, canavan disease, and 4-hydroxybutyric aciduria were also diagnosed. Prompt treatment following diagnosis led to a better outcome in a considerable number of patients. CONCLUSIONS: GC/MS with one-step metabolomics enables quick detection, accurate identification, and precise quantification of a wide range of urinary markers that may not be discovered using existing newborn screening programs. The technique is effective as a second-tier test to other established screening technologies, as well as one-step primary screening tool for a wide spectrum of IEM.

Haptoglobin2-2 phenotype is an additional risk factor of retinopathy in type 2 diabetes mellitus.

AIMS: The aim of this study was to investigate the association between haptoglobin (Hp) phenotypes and risk of the development of diabetic retinopathy (DR) in patients of type 2 diabetes mellitus. MATERIALS AND METHODS: This cross-sectional study included 45 normotensive type 2 diabetic patients (duration more than 5 years) admitted in the hospital divided into two groups (with and without DR) on the basis of fundus examination by direct ophthalmoscopy. Serum samples of all patients were subjected for Hp phenotyping by polyacrylamide gel electrophoresis. RESULTS: DR was associated significantly in diabetic patients with Hp2-2 phenotype (79.31%) than diabetic patients with Hp2-1 phenotype (43.75%) and Hp2-2 had higher odds ratio (OR) for DR in univariate analysis (OR 4.929, [95% confidence interval [CI] (1.297-18.733)], P = 0.016) and multivariate analysis (OR 7.704 [95% CI (0.887-66.945)], P = 0.064). Furthermore, Hp2-2 was associated significantly with severe forms of DR. CONCLUSION: Hp2-2 phenotype is associated with susceptibility to DR showing a graded risk relationship to the number of Hp2 alleles. Determination of Hp phenotype may be useful in the risk assessment and management of DR.