Epigenetic downregulation of desmin in gall bladder cancer reveals its potential role in disease progression.

Background & objectives: Gall bladder cancer (GBC) is a fatal neoplasm, with a globally variable incidence rates. To improve the survival rate of patients, a newer set of biomarkers needs to be discovered for its early detection and better prognosis. Our earlier studies on GBC proteomics and whole-genome methylome data revealed expression of desmin to be significantly downregulated with correlated promoter hypermethylation during gall bladder carcinogenesis. Thus, to evaluate desmin as a potential biomarker for GBC, we carried out a detailed follow up study. Methods: Methylation-specific polymerase chain reaction (MS-PCR) (n=17, GBC and n=23, non-tumour control), real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) [n=14, GBC and n=14, adjacent non-tumour (ANT)], immunohistochemistry (n=27, GBC and n=14, non-tumour) and immunoblotting (n=13, GBC and n=13, ANT) were performed in surgically removed gall bladder tissue samples. Results: MS-PCR analysis showed methylation of desmin in 88.23 per cent (15/17) gall bladder tumour samples as compared to non-tumour tissues (39.13%, 9/23). Real-time qRT-PCR analysis revealed a significant downregulation of desmin expression in GBC as compared to ANT tissue. This was further confirmed by western blot, showing reduced expression of desmin protein in GBC, as compared to non-tumour tissue. Immunohistochemical analysis also showed a decreased level of desmin i.e., more than 95 per cent (26/27) in tumour cells compared to non-tumours (35.71%, 5/14). Interpretation & conclusions: The increased frequency of desmin promoter methylation which could be responsible for its significant downregulation, indicates its potential as a candidate biomarker for GBC. This requires further validation in a large group of patients to evaluate its clinical utility.

Genotype-phenotype relationship of CCL5 in pulmonary tuberculosis infection in Sahariya tribe: A pilot study.

BACKGROUND & OBJECTIVES: Sahariya, a primitive tribe of Central India, has shown significantly increased incidence of pulmonary tuberculosis (PTB). Our previous study on Sahariya showed a significant association of -403G>A single nucleotide polymorphism (SNP) of CCL5 with susceptibility to PTB. Hence, this study was aimed to analyze a genotype-phenotype relationship of this disease-associated SNP to develop a potential diagnostic marker for TB in this tribe. METHODS: The present study was carried out on 70 plasma samples from Sahariya tribe, wherein the plasma CCL5 level was determined using a commercially available ELISA kit. RESULTS: The level of CCL5 decreased significantly in patients who were on therapy/completed their therapy [inactive TB patient/inactive PTB (IPTB)], particularly with AA genotype of -403G>A (P=0.046). The level, with AA genotype, was also found to gradually decrease in sputum 3+ and 1+/2+ than in sputum-negative samples. Similarly, the CCL5 level was found to be higher in sputum-positive/active TB patients than in IPTB group and healthy controls. INTERPRETATION & CONCLUSIONS: Our results suggested that the CCL5 level was influenced collectively not only by the genotypes of -403G>A SNP and bacillary load but also by the treatment. Thus, CCL5 may be considered for the development of a diagnostic marker and also as an indicator of recovery.

Global methylation profiling to identify epigenetic signature of gallbladder cancer and gallstone disease.

Promoter methylation in various tumor suppressor genes is reported to influence gallbladder carcinogenesis. Here, we aimed to identify methylation status in gallbladder cancer (GBC) by performing a comprehensive genome-wide DNA methylation profiling. The methylation status of 485,577 CpG sites were investigated using Illumina's Infinium Human Methylation 450 BeadChip array in 24 tissues (eight each of tumor, adjacent non-tumor, and gallstone). About 33,443 differentially methylated sites (DMRs) were obtained in the whole human genome, of which 24,188 (72 %) were hypermethylated and 9255 (28 %) were hypomethylated. The data also revealed that majority of the DMRs are localized on the proximal promoter region [Transcription start sites (TSS200, TSS1500) and 5' untranslated region (5'UTR)] and first exon. Exclusion of first exon detected a total of 10,123 (79 %) hypermethylated and 2703 (21 %) hypomethylated sites. Comparative analysis of the later with our differential proteomics data resulted in identification of 7 hypermethylated or down-regulated (e.g., FBN1, LPP, and SOD3) and 61 hypomethylated or up-regulated markers (e.g., HBE1, SNRPF, TPD52) for GBC. These genes could be further validated on the basis of their methylation/expression status in order to identify their utility to be used as biomarker/s for early diagnosis and management of GBC.

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

BACKGROUND & OBJECTIVES: Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD. METHODS: Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues. RESULTS: Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC ( P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression. INTERPRETATION & CONCLUSIONS: The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.

Inhibitory effects of neem seed oil and its extract on various direct acting and activation-dependant mutagens-induced bacterial mutagenesis.

CONTEXT: Azadirachta indica A. Juss (Meliaceae), commonly called neem is a plant native to the Indian sub-continent. Neem oil extracted from the seeds of neem tree has shown promising medicinal properties. OBJECTIVE: To investigate the possible anti-mutagenic activity of neem seed oil (NO) and its dimethylsulfoxide (DMSO) extract (NDE) on the mutagenicity induced by various direct acting and activation-dependant mutagens. MATERIALS AND METHODS: The possible anti-mutagenic activity of NO (100-10,000 µg/plate) and NDE (0.1-1000 µg/plate) as well as the mechanism of anti-mutagenic activity was studied in an in vitro Ames Salmonella/microsome assay. RESULTS: NSO and NDE inhibited the mutagenic activity of methyl glyoxal (MG), in which case the extent of inhibition ranged from 65 to 77% and against 4-nitroquinoline-N-oxide (NQNO); it showed a 48-87% inhibition in the non-toxic doses. Similar response of NSO and NDE was seen against the activation-dependant mutagens aflatoxin B1 (AFB1, 48-88%), benzo(a)pyrene (B(a)P, 31-85%), cyclophosphamide (CP, 66-71%), 20-methylcholanthrane (20-MC, 37-83%) and acridine orange (AO, 39-72%) in the non-toxic doses. Mechanism-based studies indicated that NDE exhibits better anti-mutagenic activity in the pre- and simultaneous-treatment protocol against MG, suggesting that one or several active phytochemicals present in the extract covalently bind with the mutagen and prevent its interaction with the genome. DISCUSSION AND CONCLUSION: These findings demonstrate that neem oil is capable of attenuating the mutagenic activity of various direct acting and activation-dependant mutagens.

Analysis of autosomal dominant spinocerebellar ataxia type 1 in an extended family of central India.

BACKGROUND: Spinocerebeller ataxia type 1 (SCA1) is a specific type of ataxia among a group of inherited diseases of the central nervous system. In SCA1, genetic defects lead to impairment of specific nerve fibers carrying messages to and from the brain, resulting in the degeneration of the cerebellum, the coordination center of the brain. We investigated 24 members of an extended family in Gwalior city, India, some of which were earlier clinically diagnosed to be suffering from yet unconfirmed type of SCA neurodegenerative disorder. MATERIALS AND METHODS: All the family members from each age group were screened clinically and the characteristics of those resembling with ataxia were recorded for diagnosis by MRI. The confirmed patients of the family were genetically tested by PCR based molecular testing to identify the type of SCA (i.e., SCA 1, 2, 3, 4, 6 or 7). Family tree of the disease inheritance was constructed by pedigree based method. RESULT AND CONCLUSION: We found the clinical (symptoms and MRI) and genetic (Pedigree and PCR) results to be correlated. The PCR result revealed the disease to be of SCA 1 type being inherited in the family.

Novel genetic polymorphisms identified in the clinical isolates of Mycobacterium tuberculosis PE_PGRS33 gene modulate cytokines expression and promotes survival in macrophages.

OBJECTIVE: PE_PGRS33 is a member of multi-gene family restricted to pathogenic Mycobacteria and their functions remain elusive. PE_PGRS33 is highly polymorphic in nature to evade host's immune response. Therefore, we investigated PE_PGRS33 gene polymorphisms in clinical isolates and functional characterization using in vitro experiments. METHODS: A total of 103 clinical isolates were recruited. Genomic DNA was extracted, PE_PGRS33 gene amplification, sequencing. Afterward, we have cloned, expressed PE_PGRS33 wild type and three polymorphic alleles in M. smegmatis. Further, performed in vitro stresses assays, THP-1 differentiated macrophage infection assays followed by quantification of cytokine expression. RESULTS: We have identified nine novel polymorphisms and also demonstrated that in comparison to M. smegmatis expressing wild type/Mut_alleles displayed altered in growth kinetics and colony morphology. M. smegmatis harbouring Mut_allele3 survived better under oxidative, acidic stress and were resistant to lysozyme treatment. qRT-PCR of cytokines TNF-α, IL-12 and IL-10 after infection with recombinant M. smegmatis showed two Mut_allele (Mut_allele1 and Mut_allele3) induced higher expression of TNF-α, IL-12, while IL-10 expression was decreased in both mutant alleles as compared to wild type PE_PGRS33 at each experimental time point. CONCLUSION: Results of our functional study suggest that PE_PGRS33 gene polymorphisms aid in the survival or persistence of M. tuberculosis and differentially modulate the expression of various cytokines. Overall this study suggests that Mtb clinical strains harbouring different PE_PGRS33 alleles could act as a virulence determinant by differentially regulating pathways essential for the pathogen's ability to adapt inside the host.

Comprehensive proteomic analysis of human bile.

Bile serves diverse functions from metabolism to transport. In addition to acids and salts, bile is composed of proteins secreted or shed by the hepatobiliary system. Although there have been previous efforts to catalog biliary proteins, an in-depth analysis of the bile proteome has not yet been reported. We carried out fractionation of non-cancerous bile samples using a multipronged approach (SDS-PAGE, SCX and OFFGEL) followed by MS analysis on an LTQ-Orbitrap Velos mass spectrometer using high resolution at both MS and MS/MS levels. We identified 2552 proteins - the largest number of proteins reported in human bile till date. To our knowledge, there are no previous studies employing high-resolution MS reporting a more detailed catalog of any body fluid proteome in a single study. We propose that extensive fractionation coupled to high-resolution MS can be used as a standard methodology for in-depth characterization of any body fluid. This catalog should serve as a baseline for the future studies aimed at discovering biomarkers from bile in gallbladder, hepatic, and biliary cancers.

Clinical epidemiology of gallbladder cancer in North-Central India and association of immunological markers, NLR, MLR and PLR in the diagnostic/prognostic prediction of GBC.

INTRODUCTION: The incidence of Gallbladder Cancer (GBC) is found to be increasing in the rural populations of north-central India. Role of multiple demographic factors, including poor socio-economic conditions, illiteracy and miserable primary healthcare services appear to be significant factors for this increase. Here, we aim to assess the present status of GBC in north-central India and evaluate the role of immunological markers in its management. METHODS: A total of 1845 cases of different Gallbladder diseases, including GBC, from rural and urban areas both, registered at CHRI, Gwalior during 2009-2014 and 2018 were included in this study. The demographic and clinical information of the patients were analysed using various statistical tests. RESULTS: Of all the cases (1845) included in this study, 1125 (60.97%) were diagnosed with GBC, of which, 707 (62.84%) were from rural background and 418 (37.15%) from urban settings. Mean age for GBC cases for both male and female was about 53.49 years. Females were more affected, being 70.37%, while male patients were only 29.63%. The pathological investigations showed elevated levels of total bilirubin and liver function enzymes both. The NLR, PLR and MLR were found to be significantly associated with different clinical parameters as well as OS. CONCLUSION: We infer that the growing trend of GBC, particularly in rural areas, in north-central India is primarily associated with the lack of awareness, inadequate medical support and poor socio-economic conditions. Evaluation of haematological markers may help in the predictive diagnosis/ prognosis and or management of GBC cases in the studied population.

COLIA1 + 1245 G > T Sp1 Binding Site Polymorphism is Not Associated with ACL Injury Risks Among Indian Athletes.

OBJECTIVE: Type I collagen polypeptides contribute significantly to the structural composition of ligament tissue matrix. Since anterior cruciate ligament (ACL) tears account for roughly 50% of all knee injuries in sports, the objective of the study was to investigate association of Sp1-transcription factor binding site polymorphism COLIA1 Sp1 + 1245 G > T with ACL injury risk among Indian athletes. METHODS: A total of 166 athletes (90 with ACL tears and 76 as control) were recruited and were genotyped for COLIA1 Sp1 + 1245 G > T polymorphism using allele-specific PCR (AS-PCR) method. RESULT: Both the groups were matched for nature of sports, training regimen, and other demographic characteristics. We observed no significant difference between ACL cases and control group in GT or TT genotype frequency distribution (p = 0.967) and T-allele frequency distribution (p = 0.861) for COLIA1 Sp1 + 1245 G > T polymorphism. Also, the three models of inheritance of minor allele failed to show any statistical significance in the present study. CONCLUSION: COLIA1 Sp1 + 1245 G > T polymorphism has been studied in relation to many connective tissue pathologies. This is probably the first study to investigate the association of collagen protein genes with ACL injury risk on Indian athletes. Further studies with more SNPs in genes encoding fibril-forming collagen and large sample sizes are necessary to fully understand the genetic link to ACL injuries among athletes.

VEGFA Promoter Polymorphisms rs699947 and rs35569394 Are Associated With the Risk of Anterior Cruciate Ligament Ruptures Among Indian Athletes: A Cross-sectional Study.

BACKGROUND: Associations of genetic variants within certain fibril-forming genes have previously been observed with anterior cruciate ligament (ACL) injuries. Evidence suggests a significant role of angiogenesis-associated cytokines in remodeling the ligament fibril matrix after mechanical loading and maintaining structural and functional integrity of the ligament. Functional polymorphisms within the vascular endothelial growth factor A (VEGFA) gene have emerged as plausible candidates owing to their role in the regulation of angiogenic responses. HYPOTHESIS: VEGFA promoter polymorphisms rs699947 and rs35569394 are associated with ACL injury risk among athletes. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: A total of 90 Indian athletes with radiologically confirmed or surgically proven isolated ACL tears and 76 matched-control athletes were selected for the present cross-sectional genetic association study. Oral mouthwash samples were collected from all the case and control athletes and genotyped for VEGFA rs699947 and rs35569394 using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The A allele (rs699947) was significantly overrepresented in the ACL group (C vs A allele: odds ratio [OR], 1.68 [95% CI, 1.08-2.60]; P = .021) (CC vs CA + AA: OR, 2.69 [95% CI, 1.37-5.26]; P = .004). There was a greater frequency of the AA genotype in the ACL group in comparison with the control group (OR, 3.38 [95% CI, 1.23-9.28]; P = .016) when only male athletes were compared. Likewise, there was a greater frequency of the I allele (rs35569394) in the ACL group (D vs I allele: OR, 1.64 [95% CI, 1.06-2.55]; P = .025) (DD vs ID + II: OR, 2.61 [95% CI, 1.31-5.21]; P = .006). The A-I haplotype was overrepresented in the ACL group compared with the control group (OR, 1.68 [95% CI, 1.08-2.60]; χ2 = 5.320; P = .021), and both the polymorphisms were found to be in complete linkage disequilibrium (r 2 = 0.929; logarithm of the odds score = 63.74; D' = 1.0). Female athletes did not show any difference in genotype or allele frequency. CONCLUSION: This is the first study to investigate the association of VEGFA promoter polymorphisms in ACL tears among Indian athletes. Increased frequencies of the A allele (rs699947) and I allele (rs35569394) were observed in the ACL group. These results suggest that sequence variants in the VEGF gene are associated with ACL injury risk among athletes. Further research with long-term follow-ups measuring VEGF expression levels during recovery is warranted to establish its role in ACL injuries and healing.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

The Indian origin of paternal haplogroup R1a1* substantiates the autochthonous origin of Brahmins and the caste system.

Many major rival models of the origin of the Hindu caste system co-exist despite extensive studies, each with associated genetic evidences. One of the major factors that has still kept the origin of the Indian caste system obscure is the unresolved question of the origin of Y-haplogroup R1a1*, at times associated with a male-mediated major genetic influx from Central Asia or Eurasia, which has contributed to the higher castes in India. Y-haplogroup R1a1* has a widespread distribution and high frequency across Eurasia, Central Asia and the Indian subcontinent, with scanty reports of its ancestral (R*, R1* and R1a*) and derived lineages (R1a1a, R1a1b and R1a1c). To resolve these issues, we screened 621 Y-chromosomes (of Brahmins occupying the upper-most caste position and schedule castes/tribals occupying the lower-most positions) with 55 Y-chromosomal binary markers and seven Y-microsatellite markers and compiled an extensive dataset of 2809 Y-chromosomes (681 Brahmins, and 2128 tribals and schedule castes) for conclusions. A peculiar observation of the highest frequency (up to 72.22%) of Y-haplogroup R1a1* in Brahmins hinted at its presence as a founder lineage for this caste group. Further, observation of R1a1* in different tribal population groups, existence of Y-haplogroup R1a* in ancestors and extended phylogenetic analyses of the pooled dataset of 530 Indians, 224 Pakistanis and 276 Central Asians and Eurasians bearing the R1a1* haplogroup supported the autochthonous origin of R1a1 lineage in India and a tribal link to Indian Brahmins. However, it is important to discover novel Y-chromosomal binary marker(s) for a higher resolution of R1a1* and confirm the present conclusions.

Genotypic diversity of Mycobacterium tuberculosis isolates from North-Central Indian population.

BACKGROUND: Different strains of Mycobacterium tuberculosis (MTB) are known to have different epidemiological and clinical characteristics. Some of them are widely distributed and associated with drug resistance, whereas others are locally predominated. Molecular epidemiological investigations have always been beneficial in identifying new strains and studying their transmission dynamics. Sahariya a primitive tribe of North Madhya Pradesh, India, has already been reported to have high prevalence of tuberculosis (TB) than their non-tribal neighbours. However, the information about MTB genotypes prevalent in Sahariya tribe and their non-tribal neighbours is not available. METHODS: A total of 214 clinical isolates representing Sahariya tribe and non-tribes were analyzed by spoligotyping and MIRU-VNTR typing. RESULTS: The EAI3_IND/SIT11 genotype was observed as major genotype in Sahariya tribe followed by CAS1_Delhi/SIT26 genotype. A 3.04 fold higher risk of getting TB with EAI3_IND/SIT11 genotype was observed in Sahariya as compared to the non-tribal population. The EAI_IND/SIT11 genotype also found to have more number of MDR-TB cases in Sahariya as well as true and possible transmission links. In Sahariya tribe, 3 clusters (6 isolates) reflected true transmission links, whereas 8 clusters consisted of 26 isolates revealed possible transmission links within the same geographical location or nearby houses. CONCLUSION: The present study highlighted the predominance of EAI3_IND/SIT11 genotype in Sahariya tribe followed by CAS1_Delhi/SIT26 genotype. Combined approach of MIRU-VNTR typing and spoligotyping was observed more favourable in discrimination of MTB genotypes. Further, longitudinal studies using whole genome sequencing can provide more insights into genetic diversity, drug resistance and transmission dynamics of these prevalent genotypes.

Epigenetic Downregulation of PTEN in Gallbladder Cancer.

PURPOSE: Gallbladder cancer (GBC) is becoming a global health problem. Phosphatase and tensin homolog (PTEN), also known as guardian gene of cancer, encodes a protein, which dephosphorylates phosphatidylinositol (3,4,5) triphosphate {PtdIns (3,4,5) P3 } into phosphatidylinositol (4,5) diphosphate {PtdIns (4,5) P2} that results in the inhibition of AKT/PKB signaling pathway. PTEN plays a key role in the pathogenesis of various cancers. However, its role in GBC is still obscure. The present study was aimed to identify the epigenetic role of PTEN in GBC and GSD. METHODS: PTEN promoter methylation was studied by methylation-specific PCR in 50 GBC and 30 GSD tissues. Transcript level expression of PTEN was analyzed by reverse transcriptase PCR and quantitative PCR in 20 GBC and 20 GSD tissue samples. Immunohistochemistry was performed on tissue microarrays of 136 GBC samples to correlate the methylation pattern with the pattern of in situ expression of PTEN protein. Student's t test was performed to test the significant level of difference between case and control samples. Values showing p ≤ 0.05 were considered significant. RESULTS: MS PCR showed PTEN promoter methylation in 30% (15/50) GBC (p = 0.0486) and 22.86% (8/30) GSD (p = 0.0530) cases. RT-PCR and qPCR revealed downregulation of PTEN in advanced GBC cases (p < 0.0001), but, not in GSD (p = 0.901). Immunohistochemistry of GBC tissue microarray scored 1+ in 20.29% (p = 0.0028) and zero or negative in 50% (p < 0.0001) GBC cores. CONCLUSIONS: Thus, PTEN may be considered as a useful biomarker for the management of GBC, however, needs larger sample size to validate it further.

Telomere length variation and expression analysis of shelterin complex genes during gallbladder carcinogenesis.

BACKGROUND: Telomeres, which are bound with shelterin protein complex, play an important role in maintaining genomic stability and its dysfunction may lead to carcinogenesis. Here, we aimed to analyze whether shelterin complex gene expression and telomere length variation, play any role in gallbladder carcinogenesis. METHODS: Telomere length analysis was carried out by monochrome multiplex qPCR, whereas expression analysis of shelterin genes was carried out using RT-qPCR. Statistical analysis was carried out using SigmaPlot 11 software. RESULTS: We found significantly reduced telomere length in tumor tissues, and this reduction was seen in both, tumors with or without gallstones in comparison to adjacent non tumor and gallstone (chronic calculous cholecystitis: Inflamed) tissues. Inflamed tissues showed increased telomere length as compared to both adjacent non tumor and tumor tissues. Expression analysis of five shelterin genes showed significant downregulation of TERF1, POT1, and TINF2 genes in inflamed tissues as compared to non tumor and tumor tissues. POT1 was also found to be significantly upregulated in tumor tissues and specifically in tumor tissues with gallstones compared to inflamed tissues. CONCLUSION: This study, thus, suggests that, gallstone does not affect telomere length and even after having increased telomere length, decreased expression of some shelterin genes in inflamed tissue might cause telomeres to cap improperly, possibly leading to telomere dysfunction and further, gallbladder carcinogenesis. Also, increased expression of POT1 in tumor tissues with gallstones could act as a diagnostic marker in patients with gallstones.

Epigenetic regulation in gallbladder cancer: Promoter methylation profiling as emergent novel biomarkers.

DNA methylation, once considered to rule the sex determination in Mary Lyon's hypothesis, has now reached the epicenter of human diseases, from monogenic (e.g. Prader Willi syndrome, Angelman syndromes and Beckwith-Wiedemann syndrome) to polygenic diseases, like cancer. Technological developments from gold standard to high throughput technologies have made tremendous advancement to define the epigenetic mechanism of cancer. Gallbladder cancer (GBC) is a fatal health issue affecting mostly the middle-aged women, whose survival rate is very low due to late symptomatic diagnosis. DNA methylation has become one of the key molecular mechanisms in the tumorigenesis of gallbladder. Various molecules have been reported to be epigenetically altered in GBC. In this review, we have discussed the classes of epigenetics, an overview of DNA methylation, technological approaches for its study, profile of methylated genes, their likely roles in GBC, future prospects of biomarker development and other discovery approaches, including therapeutics.

Non-coding RNAs as emerging molecular targets of gallbladder cancer.

Gallbladder cancer is one of the most common cancers of biliary tract with aggressive pathophysiology, now emerging as a global health issue. Although minority of gallbladder cancer patients could receive such curative resection due to late diagnosis, this increases the survival rate. Lack of potential target molecule (s) for early diagnosis, better prognosis and effective therapy of gallbladder cancer has triggered investigators to look for novel technological or high throughput approaches to identify potential biomarker for gallbladder cancer. Intervention of non-coding RNAs in gallbladder cancer has been revealed recently. Non-coding RNAs are now widely implicated in cancer. Recent reports have revealed association of non-coding RNAs (microRNAs or miRNAs and long non-coding RNAs or lncRNAs) with gallbladder cancer. Here, we present an updated overview on the biogenesis, mechanism of action, role of non-coding RNAs, the identified cellular functions in gallbladder tumorigenesis, their prognostic & therapeutic potentials (efficacies) and future significance in developing effective biomarker(s), in future, for gallbladder.

Epigenetic profiling of gallbladder cancer and gall stone diseases: Evaluation of role of tumour associated genes.

BACKGROUND: As on today, the global mortality rate of gallbladder cancer is still very high. Both genetic and epigenetic alterations play pivotal roles in the development of cancer. We selected seven tumour associated genes, implicated in other cancers, to assess their methylation status in gallbladder cancer and gallstone diseases. AIM OF STUDY: To study the promoter methylation of certain tumour associated genes in the molecular pathogenesis of gallbladder cancer and gall stone diseases. MATERIALS AND METHODS: Methylation specific PCR for seven tumour associated genes, viz., MASPIN, 14-3-3 sigma gene, THBS1, FLNC, HLTF, COX-2 and SOCS1, was performed in 50 gallbladder cancer (GBC), 30 gall stone diseases (GSD) and their respective adjacent control tissues. Semi-quantitative PCR and immunohistochemistry was carried out to check the expression level. Student's t-test was carried out to compare the differences in the methylation and expression patterns between cases and control tissues. RESULTS: We observed methylation of CpG islands in seven of the studied markers, but, the frequency of methylation was found varying among different samples. Of them, 14-33 sigma showed methylation in 45 GBC (90%; p=0.0001) and 25 GSD (86.66%; p=0.001), MASPIN in 35 GBC (70%; p=0.0008) and 18 GSD (51.43%; p=0.040), FLNC in 16 GBC (32%; p=0.0044) and 9 GSD (25.71%; p=ns), THBS1 in 26 GBC (52%; p=0.0009) and 10 GSD (28.57%; p=0.0505), HLTF in 8 GBC (16%; p=ns) and 2 GSD (5.71%; p=ns), COX2 in 10 GBC (20%; p=ns) and 6 GSD (17.14%; p=ns) and SOCS-1 in 3 GBC samples only (6%; p=ns), but not in GSD. Semi-quantitative PCR revealed down regulation in MASPIN, 14-3-3 sigma, THBS1, HLTF, COX2 and SOCS1 in advanced gallbladder cases. Immunohistochemistry further confirmed the down-regulation of SOCS1 in GBC. CONCLUSION: The present study infers that accumulation of epigenetic alterations increases poor prognosis of GBC patients. Out of seven genes, MASPIN and THBS1 play key epigenetic role in GBC, but not in GSD. The reason for downregulation of SOCS1 only in GBC, and unaltered expression of 14-3-3 sigma protein in all the GBC and GSD tissue samples is not clear. Further investigation on the expression pattern of these genes in GBC cell lines may elucidate their likely functional role in in association with gallbladder cancer.

Dysfunction of subtelomeric methylation and telomere length in gallstone disease and gallbladder cancer patients of North Central India.

BACKGROUND: Telomeres play an important role in cancer progression. Recently it has been shown that subtelomeric methylation negatively regulates telomere length in various diseases, including cancers. Here, we evaluated the influence of subtelomeric methylation in telomere dysfunction in gallbladder cancer (GBC), and whether this dysfunction is affected by the presence of gallstones. METHODS: Relative telomere length and subtelomeric methylation levels were assessed using monochrome multiplex quantitative polymerase chain reaction and bisulfite sequencing, respectively, in different gallbladder tissue types including different grades of GBC, gallstones and adjacent non-tumor. RESULTS: We found telomere length to shorten significantly in overall GBC, but specifically in early grade cancer. We also found D4Z4 and DNF92 subtelomeric sequences to be hypermethylated and hypomethylated, respectively, in GBC; however, their methylation levels differed significantly, only in early grade cancer. We could not find any specific correlation between subtelomeric methylation and telomere length in GBC. Interestingly, telomere length and subtelomeric methylation differed significantly in GBC without gallstones but not in GBC with gallstones. CONCLUSIONS: This study, thus, suggests that telomere dysfunction and changes in methylation levels may occur earlier in the progression of GBC, while the presence of gallstones may have no influence on telomere length as well as on methylation levels.