A novel Tetra-primer ARMS-PCR based assay for genotyping SNP rs12303764(G/T) of human Unc-51 like kinase 1 gene.

Various case-control studies have shown association of single nucleotide polymorphism rs12303764(G/T) in ULK1 with crohn's disease. The techniques used in these studies were time consuming, complicated and require sophisticated/expensive instruments. Therefore, in order to overcome these problems, we have developed a new, rapid and cost effective Tetra-primer ARMS-PCR assay to genotype single nucleotide polymorphism rs12303764(G/T) of ULK1 gene. We manually designed allele specific primers. DNA fragment amplified using outer primers was sequenced to obtain samples with known genotypes (GG, GT and TT) for further use in the development of T-ARMS-PCR assay. Amplification conditions were optimized for parameters; annealing temperature, Taq DNA polymerase and primers. The developed T-ARMS-PCR assay was applied to genotype one hundred samples from healthy individuals. Genotyping results of 10 DNA samples from healthy individuals for rs12303764(G/T) by T-ARMS-PCR assay and sequencing were concordant. The newly developed assay was further applied to genotype samples from 100 healthy individuals of North Indian origin. Genotype frequencies were 9, 34 and 57 % for GG, GT and TT, respectively. Allele frequencies were 0.26 and 0.74 for G and T, respectively. The allele frequencies were in Hardy-Weinberg's equilibrium (p = 0.2443). T-ARMS-PCR assay developed in our laboratory for genotyping rs12303764 (G/T) of ULK1 gene is time saving and cost-effective as compared to the available methods. Furthermore, this is the first study reporting allelic and genotype frequencies of ULK1 rs12303764 (G/T) variants in North Indian population.

Investigating regulatory signatures of human autophagy related gene 5 (ATG5) through functional in silico analysis.

Autophagy is an essential, homeostatic process which removes damaged cellular proteins and organelles for cellular renewal. ATG5, a part of E3 ubiquitin ligase-like complex (Atg12-Atg5/Atg16L1), is a key regulator involved in autophagosome formation - a crucial phase of autophagy. In this study, we used different in silico methods for comprehensive analysis of ATG5 to investigate its less explored regulatory activity. We have predicted various physico-chemical parameters and two possible transmembrane models that helped in exposing its functional regions. Twenty four PTM sites and 44 TFBS were identified which could be targeted to modulate the autophagy pathway. Furthermore, LD analysis identified 3 blocks of genotyped SNPs and 2 deleterious nsSNPs that may have damaging impact on protein function and thus could be employed for carrying genome-wide association studies. In conclusion, the information obtained in this study could be helpful for better understanding of regulatory roles of ATG5 and provides a base for its implication in population-based studies.

Liver X Receptor-α polymorphisms (rs11039155 and rs2279238) are associated with susceptibility to vitiligo.

Vitiligo is a complex genetic skin depigmentation disorder caused by the destruction of melanocyte from the lesional site. Liver X Receptor-α (LXR-α) expression is upregulated in the melanocytes from perilesional skin as compared to the normal skin of vitiligo patient suggesting its involvement in vitiligo pathogenesis. Polymorphisms in LXR-α have been associated with several diseases including cardiovascular disease, polycystic ovary syndrome, cancer, inflammatory bowel disease and diabetes. In this study, for the first time, we have investigated the association of LXR-α gene polymorphisms and risk of vitiligo. Sixty six vitiligo patients and 75 matched healthy control subjects who did not have any history of vitiligo or any other autoimmune disorder were recruited. The DNA isolated from patients and healthy controls was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for both rs11039155 (- 6 G > A) and rs2279238 (+ 1257 C > T) variants. Our data suggest significant association between the LXR-α gene polymorphisms and vitiligo susceptibility (rs11039155: odds ratio (OR) = 1.99, 95% CI = 1.07-3.71, p = 0.03; rs2279238: OR = 1.70, 95% CI = 1.06-2.73, p = 0.027). Our results provide an evidence that the LXR-α - 6A and + 1257T alleles contribute to risk of vitiligo in North Indian population and highlight the importance of this gene in the vitiligo pathogenesis.

Unc-51 like kinase 1 (ULK1) in silico analysis for biomarker identification: a vital component of autophagy.

Autophagy is a degradation pathway involving lysosomal machinery for degradation of damaged organelles like the endoplasmic reticulum and mitochondria into their building blocks to maintain homeostasis within the cell. ULK1, a serine/threonine kinase, is conserved across species, from yeasts to mammals, and plays a central role in autophagy pathway. It receives signals from upstream modulators such as TIP60, mTOR and AMPK and relays them to its downstream substrates like Ambra1 and ZIP kinase. The activity of this complex is regulated through protein-protein interactions and post-translational modifications. Applying in silico analysis we identified (i) conserved patterns of ULK1 that showed its evolutionary relationship between the species which were closely related in a family compared to others. (ii) A total of 23 TFBS distributed throughout ULK1 and nuclear factor (erythroid-derived) 2 (NFE2) is of utmost significance because of its high importance rate. NEF2 has already been shown experimentally to play a role in the autophagy pathway. Most of these were of zinc coordinating class and we suggest that this information could be utilized to modulate this pathway by modifying interactions of these TFs with ULK1. (iii) CATTT haplotype was prominently found with frequency 0.774 in the studied population and nsSNPs which could have harmful effect on ULK1 protein and these could further be tested. (iv) A total of 83 phosphorylation sites were identified; 26 are already known and 57 are new that include one at tyrosine residue which could further be studied for its involvement in ULK1 regulation and hence autophagy. Furthermore, 4 palmitoylation sites at positions 426, 927, 1003 and 1049 were also found which could further be studied for protein-protein interactions as well as in trafficking.