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.

Characterization of the CYP21A2 Gene Mutations in Children with Classic Congenital Adrenal Hyperplasia.

OBJECTIVE: To characterize the CYP21A2 gene mutations in children with classic congenital adrenal hyperplasia (CAH). METHODS: A prospective, cross-sectional study was conducted on 24 children with classic CAH. Molecular characterization of the CYP21A2 gene was carried out by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), or clinical exome sequencing. Another 21 previously mutation-proven CAH patients were also included and a combined result was drawn. RESULTS: Out of 45 children, pathogenic variants in the CYP21A2 gene were identified in 43 patients (95.5%). Homozygous, probable compound heterozygous, and heterozygous variants were seen in 69%, 22%, and 18% of patients, respectively. The most common variant was c.293-13C/A>G (33%), followed by deletion/duplication (24%), and c.955C>T (p.Gln319Ter) (21%), similar to previous Indian studies. Allelic frequencies of c.332_339del and c.518 T>A (p.Ile173Asn) were 9% and 4%, respectively. Less common variants were c.923dupT (p.Leu308PhefsTer6), c.92C>T (p.Pro31Leu), c.1069C>T (p.Arg357Trp), c.1267G>C (p.Gly423Arg), and c.710_719delins (p.Ile237_Met240delinsAsnGluGluLys). A good genotype-phenotype correlation was observed; only p.Pro31Leu and p.Ile173Asn variants showed discordance. The diagnostic yield of Sanger sequencing alone, Sanger sequencing with MLPA, and clinical exome alone was 85%, 100%, and 100%, respectively. CONCLUSIONS: All children, except two, diagnosed clinically as classic CAH, showed pathogenic variants in the CYP21A2 gene; the most common variant was c.293-13 C/A>G. The results suggest a broad mutation spectrum in the authors' single-center cohort of children with CAH. Clinical exome sequencing is the preferred stand-alone method for molecular diagnosis of CAH.

First Report of the 3'-Untranslated Region +1506 (A>C) [NM_000518.5: c.*32A>C] mutation on the ß-Globin Gene in the Indian Population.

The 3'-untranslated region (3'-UTR) is well known to be associated with the post-transcriptional regulation, because of the presence of important sequences that influence the fate of mRNA, and thus, in protein synthesis. The present study describes a point mutation on the ß-globin 3'-UTR, +1506 (A>C) (HBB: c.*32A>C) in an Indian family during prenatal diagnosis (PND) screening of an at-risk couple. The members of the family heterozygous for this mutation presented with a typical ß-thalassemia (ß-thal) phenotype. The haplotype analysis of the ß-globin gene cluster was determined for this mutation and observed to be linked with haplotype [- + - + + + +]. Common α-globin gene deletions, triplication, and the Xmnl polymorphism, were also looked for and found to be absent in the family. The identified HBB: c.*32A>C mutation is located in the first adenylate uridylate (AU) motif of the four AU motifs situated in the 3'-UTR region of the ß-globin gene. Bioinformatics analysis revealed binding of two miRNAs, hsa-miR-451a and hsa-miR-3914, at the mutation position, possibly influencing the mRNA stability by recruiting RNA binding proteins. This is the third publication reporting the 3'-UTR +1506 (A>C) mutation worldwide and the first report of the existence of this mutation in the Indian population, emphasizing the high heterogeneity of this population.

Intricate Regulation of Phosphoenolpyruvate Carboxykinase (PEPCK) Isoforms in Normal Physiology and Disease.

BACKGROUND: The phosphoenolpyruvate carboxykinase (PEPCK) isoforms are considered as rate-limiting enzymes for gluconeogenesis and glyceroneogenesis pathways. PEPCK exhibits several interesting features such as a) organelle-specific isoforms (cytosolic and a mitochondrial) in vertebrate clade, b) tissue-specific expression of isoforms and c) organism-specific requirement of ATP or GTP as a cofactor. In higher organisms, PEPCK isoforms are intricately regulated and activated through several physiological and pathological stimuli such as corticoids, hormones, nutrient starvation and hypoxia. Isoform-specific transcriptional/translational regulation and their interplay in maintaining glucose homeostasis remain to be fully understood. Mounting evidence indicates the significant involvement of PEPCK isoforms in physiological processes (development and longevity) and in the progression of a variety of diseases (metabolic disorders, cancer, Smith-Magenis syndrome). OBJECTIVE: The present systematic review aimed to assimilate existing knowledge of transcriptional and translational regulation of PEPCK isoforms derived from cell, animal and clinical models. CONCLUSION: Based on current knowledge and extensive bioinformatics analysis, in this review we have provided a comparative (epi)genetic understanding of PCK1 and PCK2 genes encompassing regulatory elements, disease-associated polymorphisms, copy number variations, regulatory miRNAs and CpG densities. We have also discussed various exogenous and endogenous modulators of PEPCK isoforms and their signaling mechanisms. A comprehensive review of existing knowledge of PEPCK regulation and function may enable identification of the underlying gaps to design new pharmacological strategies and interventions for the diseases associated with gluconeogenesis.

Comparative analysis of copy number variations in ulcerative colitis associated and sporadic colorectal neoplasia.

BACKGROUND: The incidence of and mortality from colorectal cancers (CRC) can be reduced by early detection. Currently there is a lack of established markers to detect early neoplastic changes. We aimed to identify the copy number variations (CNVs) and the associated genes which could be potential markers for the detection of neoplasia in both ulcerative colitis-associated neoplasia (UC-CRN) and sporadic colorectal neoplasia (S-CRN). METHODS: We employed array comparative genome hybridization (aCGH) to identify CNVs in tissue samples of UC nonprogressor, progressor and sporadic CRC. Select genes within these CNV regions as a panel of markers were validated using quantitative real time PCR (qRT-PCR) method along with the microsatellite instability (MSI) in an independent cohort of samples. Immunohistochemistry (IHC) analysis was also performed. RESULTS: Integrated analysis showed 10 overlapping CNV regions between UC-Progressor and S-CRN, with the 8q and 12p regions showing greater overlap. The qRT-PCR based panel of MYC, MYCN, CCND1, CCND2, EGFR and FNDC3A was successful in detecting neoplasia with an overall accuracy of 54% in S-CRN compared to that of 29% in UC neoplastic samples. IHC study showed that p53 and CCND1 were significantly overexpressed with an increasing frequency from pre-neoplastic to neoplastic stages. EGFR and AMACR were expressed only in the neoplastic conditions. CONCLUSION: CNVs that are common and unique to both UC-associated and sporadic colorectal neoplasm could be the key players driving carcinogenesis. Comparative analysis of CNVs provides testable driver aberrations but needs further evaluation in larger cohorts of samples. These markers may help in developing more effective neoplasia-detection strategies during screening and surveillance programs.

Context Dependent Regulation of Human Phosphoenolpyruvate Carboxykinase Isoforms by DNA Promoter Methylation and RNA Stability.

Cytoplasmic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M) regulate hepatic gluconeogenesis to control systemic glucose homeostasis. Transcriptional and post-transcriptional mechanisms may govern synthesis, maintenance and cooperative function of compartmentalized PEPCK enzymes. In a comparative analysis, we show that tumor cells consistently transcribe and translate higher levels of enzymatically active PEPCK-C than PEPCK-M and both the isoforms were present at lower levels in normal fibroblasts. Unlike in PEPCK-M, absence of glucose reduced the PEPCK-C mRNA and protein levels only in HepG2 cells. Interestingly, isoflavone genistein significantly increased PEPCK-C mRNA and protein levels in normal fibroblasts indicating cell type specific control mechanisms. Genistein also significantly affected RNA stability of PEPCK-C but not PEPCK-M in HepG2 cells. This was due to the conserved and functional mRNA destabilizing AU rich sequences at the 3'-UTR region of PEPCK-C gene and was confirmed by luciferase reporter assays suggesting that glucose deprivation and genistein targets these sequences for mRNA degradation in HepG2 cells but not in fibroblasts. Analysis of promoter methylation by luciferase reporter assays and bisulfite DNA sequencing suggested that PEPCK-C but not PEPCK-M promoter was activated by 5-aza-2-deoxycytidine by inducing cytosine demethylation at the specific CpG dinucleotides of 5'-UTR region. Taken together, our data suggests stable PEPCK-M activity and identifies intricate relationship between (1) mRNA stability and (2) promoter DNA methylation as two mechanisms of gene expression that distinguishes PEPCK-C and PEPCK-M enzyme activities in a context and cell type dependent manner during gluconeogenesis. J. Cell. Biochem. 117: 2506-2520, 2016. © 2016 Wiley Periodicals, Inc.

Population specific impact of genetic variants in KCNJ11 gene to type 2 diabetes: a case-control and meta-analysis study.

BACKGROUND AND OBJECTIVES: Potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11) gene have a key role in insulin secretion and is of substantial interest as a candidate gene for type 2 diabetes (T2D). The current work was performed to delineate the genetic influence of KCNJ11 polymorphisms on risk of T2D in South Indian population through case-control association study along with systematic review and meta-analysis. METHODS: A case-control study of 400 T2D cases and controls of South Indian origin were performed to analyze the association of KCNJ11 polymorphisms (rs5219, rs5215, rs41282930, rs1800467) and copy number variations (CNV) on the risk of T2D. In addition a systematic review and meta-analysis for KCNJ11 rs5219 was conducted in 3,831 cases and 3,543 controls from 5 published reports from South-Asian population by searching various databases. Odds ratio with 95% confidence interval (CI) was used to assess the association strength. Cochran's Q, I2 statistics were used to study heterogeneity between the eligible studies. RESULTS: KCNJ11 rs5215, C-G-C-C haplotype and two loci analysis (rs5219 vs rs1800467) showed a significant association with T2D but CNV analysis did not show significant variation between T2D cases and control subjects. Lower age of disease onset (P = 0.04) and higher body mass index (BMI) (P = 0.04) were associated with rs5219 TT genotype in T2D patients. The meta-analysis of KCNJ11 rs5219 on South Asian population showed no association on susceptibility to T2D with an overall pooled OR = 0.98, 95% CI = 0.83-1.16. Stratification analysis showed East Asian population and global population were associated with T2D when compared to South Asians. CONCLUSION: KCNJ11 rs5219 is not independently associated with T2D in South-Indian population and our meta-analysis suggests that KCNJ11 polymorphism (rs5219) is associated with risk of T2D in East Asian population and global population but this outcome could not be replicated in South Asian sub groups.