Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). METHODS: A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. RESULTS: We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). CONCLUSION: The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

Autophagy Gene BECN1 Intronic Variant rs9890617 Predisposes Individuals to Hepatitis B Virus Infection.

Beclin 1 protein encoded by the BECN1 gene plays a critical role in the autophagy pathway which is utilized by the Hepatitis B virus (HBV) for its replication. HBV is known for the subversion of the host's autophagy process for its multiplication. The aim of this study was to determine the role of BECN1 intronic variants in HBV susceptibility. Intronic region variant rs9890617 was analyzed using Human splicing finder v3.1 and was found to alter splicing signals. A total of 712 individuals (494 HBV infected and 218 healthy controls) were recruited in the study and genotyped by applying Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis revealed that the mutant allele T of rs9890617 was significantly associated with the overall disease risk in the allelic model (OR 1.41; 95%CI 1.00-1.99, p = 0.04). On stratifying the data based on the different stages of HBV infection, the mutant genotype showed a significant association with the chronic group in allelic (OR 1.62; 95%CI 1.11-2.39, p = 0.01), dominant (OR 1.64; 95%CI 1.07-2.52, p = 0.02), and co-dominant (OR 1.55; 95%CI 1.00-2.40, p = 0.04) models. Overall, this is the first study regarding beclin 1 variant rs9890617 and we found a significant association of the mutant T allele with the genetic predisposition to HBV infection.

Autophagy related gene 5 polymorphism rs17587319 (C/G) in asthmatic patients in North Indian population.

Objective: Genetic background and environmental stimuli play an important role in asthma, which is an individual's hyper-responsiveness to these stimuli leading to airway inflammation. Autophagy Related Gene 5 (ATG5) plays a critical role in the autophagy pathway and has been shown to be involved in asthma. The genetic polymorphisms in the ATG5 have been reported to predispose individuals to asthma. The role of single nucleotide polymorphism rs17587319 (C/G) of ATG5 in asthma has not been studied so far.Materials and methods: In this study, we in silico analysed rs17587319 (C/G) using web-based tools Human Splice Finder (HSF) and RegulomeDB and further a case-control study was conducted that included 187 blood samples (94 asthmatic and 93 healthy controls).Results: In silico analysis suggested alteration of splicing signals by this intronic variant. The samples were genotyped by applying the PCR-RFLP method. The MAF obtained was 0.022 and 0.043 in healthy controls and asthmatic individuals, respectively. The statistical analysis revealed no association (allelic model, OR = 2.02, 95%CI = 0.59-6.83, p = 0.25; co-dominant model, OR = 2.06, 95%CI = 0.6-7.12, p = 0.24) of rs17587319 (C/G) with the susceptibility to asthma in the north Indian population.Conclusions: In conclusion, rs17587319 (C/G) of ATG5 does not predispose individuals to asthma in our part of the world. Further studies are needed including more number of samples to ascertain the role of this polymorphism in asthma.

Identification of molecular signatures and molecular dynamics simulation of highly deleterious missense variants of key autophagy regulator beclin 1: a computational based approach.

Beclin 1 is a key autophagy regulator that also plays significant roles in other intracellular processes such as vacuolar protein sorting. Beclin 1 protein functions as a scaffold in the formation of a multiprotein assemblage during autophagy. Beclin 1 is involved in various diseases such as cancers, neurodegenerative and autophagy-related disorders. In this study, we have used various in silico tools to scan beclin 1 at the molecular level to find its molecular signatures. We have predicted and analysed deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of beclin 1 causing alterations in its structure and also affecting its interactions with other proteins. In total, twelve coding region deleterious variants were predicted using sequence-based tools and nine were predicted using various structure-based tools. The molecular dynamics (MD) simulations revealed an altered stability of the native structure due to the introduction of mutations. Destabilization of beclin 1 ECD domain was observed due to nsSNPs W300R and E302K. Beclin 1 deleterious nsSNPs were predicted to show significant effects on beclin 1 interactions with ATG14L1, UVRAG and VPS34 proteins and were also predicted to alter the protein-protein interface of beclin 1 complexes. Additionally, beclin 1 was predicted to have thirty-one potential phosphorylation and three ubiquitination sites. In conclusion, the molecular details of beclin 1 could help in the better understanding of its functioning. The study of nsSNPs and their effect on beclin 1 and its interactions might aid in understanding the basis of anomalies caused due to beclin 1 dysfunction.Communicated by Ramaswamy H. Sarma.

ATG5: A central autophagy regulator implicated in various human diseases.

Autophagy, an intracellular conserved degradative process, plays a central role in the renewal/recycling of a cell to maintain the homeostasis of nutrients and energy within the cell. ATG5, a key component of autophagy, regulates the formation of the autophagosome, a hallmark of autophagy. ATG5 binds with ATG12 and ATG16L1 resulting in E3 like ligase complex, which is necessary for autophagosome expansion. Available data suggest that ATG5 is indispensable for autophagy and has an imperative role in several essential biological processes. Moreover, ATG5 has also been demonstrated to possess autophagy-independent functions that magnify its significance and therapeutic potential. ATG5 interacts with various molecules for the execution of different processes implicated during physiological and pathological conditions. Furthermore, ATG5 genetic variants are associated with various ailments. This review discusses various autophagy-dependent and autophagy-independent roles of ATG5, highlights its various deleterious genetic variants reported until now, and various studies supporting it as a potential drug target.

The AGE-RAGE Axis and RAGE Genetics in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous group of lung diseases limiting the airflow due to narrowing of airways, chronic bronchitis and emphysema that leads to difficulties in breathing. Chronic inflammation is another important characteristic of COPD which leads to immune cell infiltration and helps in the alveolar destruction. Pathology of COPD is driven by various environmental and genetic factors. COPD is mainly associated with the inhalation of toxic agents mainly the cigarette smoke. Receptor for advanced glycation end products (RAGE) has emerged as a pattern recognition receptor and is a multiligand receptor expressed moderately in various cells, tissues and highly in the lungs throughout life. RAGE recognizes various ligands produced by cigarette smoke and its role has been implicated in the pathogenesis of COPD. RAGE ligands have been reported to accumulate in the lungs of patients with COPD. RAGE is a membrane receptor but its truncated form i.e. soluble RAGE (sRAGE) mainly functions as a contender of RAGE and inhibits various RAGE dependent cell signalling. Among the various ligands of RAGE, advanced glycation end products (AGEs) are majorly linked with COPD. Accumulated AGE triggers downstream RAGE-AGE axis in COPD. Moreover, RAGE genetics has long been known to play a vital role in the pathology of various airway diseases including COPD and this gene contains an associated locus. A reliable biomarker is needed for the management of this disease. sRAGE has an inverse correlation with the RAGE showed its importance as a valuable marker in COPD. This review is focused on the role of RAGE, sRAGE, RAGE axis and RAGE genetics in COPD.

The autophagy gene ATG16L1 (T300A) variant is associated with the risk and progression of HBV infection.

Autophagy pathway genes variants that play crucial roles in immune responses are involved in many diseases but their role in viral diseases is ill-defined. ATG16L1 gene plays a crucial role in the autophagy process. In this study, we have investigated the role of ATG16L1 variant T300A in the risk of HBV infection. rs2241880 (T300A) variant in 551 HBV infected patients (at various stages of infection) and 247 healthy controls were genotyped applying PCR-RFLP. Data analysis revealed that mutant allele G contributes to the risk of hepatitis B infection. Mutant alleles were significantly associated the HBV risk in allelic (OR = 1.31; 95%CI = 1.06-1.63, p = .01) and homozygous (OR = 1.87; 95%CI = 1.17-2.99, p = .009) models. On stratifying HBV infected individuals according to the stage of infection, a significant association was observed in asymptomatic (allelic; OR = 1.52; 95%CI = 1.10-2.09, p = .01 and homozygous; OR = 2.30; 95%CI = 1.22-4.36, p = .01) and chronic (allelic; OR = 1.36; 95%CI = 1.07-1.73, p = .01 and homozygous; OR = 2.07; 95%CI = 1.22-3.53, p = .008) stages of infection. High HBV DNA levels were associated with mutant genotype GG in asymptomatic and chronic carriers. Significantly higher ALT levels were observed in the liver cirrhosis patients with mutant genotypes. In conclusion, our data suggest that rs2241880 mutant allele carriers (allelic and homozygous models) were associated with increased risk of hepatitis B virus infection in North Indian population.

The beclin 1 interactome: Modification and roles in the pathology of autophagy-related disorders.

Beclin 1 a yeast Atg6/VPS30 orthologue has a significant role in autophagy process (Macroautophagy) and protein sorting. The function of beclin 1 depends on the interaction with several autophagy-related genes (Atgs) and other proteins during the autophagy process. The role mediated by beclin 1 is controlled by various conditions and factors. Beclin 1 is regulated at the gene and protein levels by different factors. These regulations could subsequently alter the beclin 1 induced autophagy process. Therefore, it is important to study the components of beclin 1 interactome and factors affecting its expression. Expression of this gene is differentially regulated under different conditions in different cells or tissues. So, the regulation part is important to study as beclin 1 is one of the candidate genes involved in diseases related to autophagy dysfunction. This review focuses on the functions of beclin 1, its interacting partners, regulations at gene and protein level, and the role of beclin 1 interactome in relation to various diseases along with the recent developments in the field.