Reversible male contraception by targeted inhibition of serine/threonine kinase 33.

Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.

Identification of potent pan-ephrin receptor kinase inhibitors using DNA-encoded chemistry technology.

EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1ß-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.

DNA-encoded chemical libraries yield non-covalent and non-peptidic SARS-CoV-2 main protease inhibitors.

The development of SARS-CoV-2 main protease (Mpro) inhibitors for the treatment of COVID-19 has mostly benefitted from X-ray structures and preexisting knowledge of inhibitors; however, an efficient method to generate Mpro inhibitors, which circumvents such information would be advantageous. As an alternative approach, we show here that DNA-encoded chemistry technology (DEC-Tec) can be used to discover inhibitors of Mpro. An affinity selection of a 4-billion-membered DNA-encoded chemical library (DECL) using Mpro as bait produces novel non-covalent and non-peptide-based small molecule inhibitors of Mpro with low nanomolar Ki values. Furthermore, these compounds demonstrate efficacy against mutant forms of Mpro that have shown resistance to the standard-of-care drug nirmatrelvir. Overall, this work demonstrates that DEC-Tec can efficiently generate novel and potent inhibitors without preliminary chemical or structural information.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

Conservative management of suspected fractures in men undergoing collagenase clostridium histolyticum for Peyronie's Disease is not associated with worsening of erectile function.

The optimal management of suspected penile fractures post collagenase clostridium histolyticum (CCH) remains indeterminate, with some advocating for observation and others surgical repair. To address this issue, the current study sent surveys to 158 men with Peyronie's Disease (PD) who completed four CCH injection series. The survey included the Erectile Function Domain from the International Index of Erectile Function (IIEF-EFD) and questions regarding potential symptoms of corporal rupture (hematoma, popping, and detumescence). Men were categorized as having a suspected fracture (SF+) if they reported a popping sensation or rapid detumescence. All SF(+) men were managed conservatively without surgical intervention. Results were compared statistically against baseline IIEF-EFD values and between SF(+) and SF(-) groups. The key study objective was to determine whether erectile function was negatively impacted by conservative management of suspected fractures. Of the 53 returned surveys, 45 had complete data for review. The sample was statistically representative of the broader cohort of 158 men, except being older (60.0 vs 57.1 [SD 6.0 vs 9.0], p = 0.01) with shorter durations of PD (median 9 [IQR 5, 19] mo vs 13 [IQR 8, 24], p = 0.01). Overall, 7/45 (16%) of men were defined as SF(+), with all fractures occurring within 6 weeks of CCH administration. No demographic or pathophysiologic characteristics predicted SF(+). Importantly, SF(+) men did not experience worsened erectile function compared to SF(-), with a median IIEF-EFD change of +2 vs +1, p = 0.16, respectively. Curvatures were improved to a greater degree among SF(+) men (primary: median -30 [IQR -20, -32.5] vs -15 [-5, -26], p = 0.04; composite: -35 [-25, -40] vs -25 [-7, -30], p = 0.15). We concluded that suspected penile fractures in PD men undergoing CCH may be reasonably managed without surgical intervention and portend greater improvements in curvature correction.

DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

Discovery and characterization of bromodomain 2-specific inhibitors of BRDT.

Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure-activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.

Reinforcing vasal suture technique improves sperm concentration and pregnancy rates in men undergoing vasovasostomy for vasectomy reversal.

BACKGROUND: Vasovasostomy (VV) is a well-described surgical technique with few notable modifications since microsurgical adaptation in the 1970s. Although contemporary reversal success rates are 70-90%, these most often are based on a lenient definition of >0 sperm (patency) and include only VV procedures. With stricter definitions, success rates drop >30%. To improve outcomes, a novel surgical technique (reinforcing vasal suture, ReVas) was developed, and outcomes were compared prior to and following implementation. METHODS: A prospective registry of sequential patients undergoing vasectomy reversal was queried from Jan 2014 to June 2019. The ReVas technique was implemented in Jan 2018, wherein the abdominal and testicular vasa are secured side-to-side to alleviate strain on the anastomosis. Primary outcomes were changes in sperm concentration: >0/mL, >100,000/mL, >1 million/mL, >5 million/mL, >15 million/mL, and most recent. Secondary outcome was pregnancy rate. Demographic, clinical, and select operative variables were statistically compared between ReVas (+) and (-) cohorts. RESULTS: A total of 200 men underwent reversal, of whom 169 represented first-time attempts (61 receiving the new technique) and comprise the current cohort. ReVas (+) and (-) cohorts were similar in demographic, clinical, and operative factors with the exception of operative time [longer in ReVas (+) group]. Median duration since vasectomy was 9 years, and 68.6% of men received a bilateral VV. Follow-up was significantly longer in the ReVas (-) arm (37 vs. 10 months). All primary outcomes were significantly higher in the ReVas (+) cohort, with odds ratios ranging from 5.8 to 11.1 (P<0.01 to 0.0001). Pregnancy rates within the first 2 years post reversal were also 8.1× higher in the ReVas (+) group (P=0.02). A subset of men with bilateral VV exhibited a 95% likelihood of achieving >15 million/mL in ReVas (+) men compared to 54% in ReVas (-). Multivariable analysis confirmed ReVas as an independent predictor of success. CONCLUSIONS: Implementation of the ReVas technique resulted in significantly higher sperm concentrations, which were particularly pronounced when stricter success criteria were used. Patients were also 8.1× more likely to achieve a pregnancy within the first 2 years, confirming clinical relevance. External validation is warranted.

The Etiology of Peyronie's Disease: Pathogenesis and Genetic Contributions.

INTRODUCTION: Peyronie's disease (PD) is a chronic fibrosing condition that contributes to penile deformity, curvature, and pain. Initial familial studies demonstrated potential genetic links to PD. Since that time, very few investigations have significantly advanced the science in this area. Hence, there is a large opportunity and significant need to better study the underlying genomics and pathogenesis of PD. AIM: To summarize the current genomic literature relevant to PD. METHODS: A review was performed of all PubMed-indexed literature from 1970-2018 relating to the pathophysiology and genetics of PD. Key findings were categorically summarized to include epidemiology, risk factors, inheritance patterns, chromosomal instability, genetic associations, epigenetics, differential gene expression, and preclinical models of PD. MAIN OUTCOME MEASURES: Summary of the current literature on the genetics of PD. RESULTS: PD is a common condition and has several known risk factors and comorbid disease associations. Although men with PD are believed to be genetically predisposed, there are likely several subtypes of the condition, each with varied pathophysiological disorders and contributing factors. Available data suggest that PD is associated with underlying genetic instability, including dysregulation of genes relating to fibrosis and cellular degradation, thus, resulting in abnormal plaque development and penile deformity. Preclinical models, including cell cultures and rat models, demonstrate several consistencies with PD clinical and histopathologic characteristics; however, an ideal model with spontaneous development of PD is lacking. CONCLUSION: Based on limited data, PD likely represents a heterogeneous condition, with both heritable and environmentally-driven epigenetic factors contributing to its development and progression. However, there remains a significant gap in the literature on the underlying cause and pathophysiology of the condition, suggesting a substantial need for further investigation and study. Sharma KL, Alom M, Trost L. The Etiology of Peyronie's Disease: Pathogenesis and Genetic Contributions. Sex Med Rev 2020;8:314-323.

Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile Traction Therapy in Men with Peyronie's Disease.

BACKGROUND: Previous studies of penile traction therapy (PTT) devices have demonstrated limited/no efficacy when combined with intralesional therapies for Peyronie's disease (PD). Recently, randomized data have demonstrated the efficacy of a novel PTT device, RestoreX, developed in cooperation with the Mayo Clinic, in men with PD. AIM: To assess the safety and efficacy of treatment with the RestoreX device plus collagenase Clostridium histolyticum (CCH) compared with CCH alone and CCH with other PTT devices. METHODS: A prospective registry has been maintained of all men undergoing CCH injections for PD between March 2014 and January 2019. Assessments were performed at baseline, during each series, and after completion of treatment. Those completing therapy (8 injections or sooner if satisfied) were categorized into group 1 (CCH alone), group 2 (CCH plus any PTT device other than RestoreX), or group 3 (CCH plus RestoreX). OUTCOMES: Changes in penile length, curvature, and subjective perception and the occurrence of adverse events. RESULTS: Of 287 men with data on PTT use, 113 had completed therapy with all objective data available and compose the current cohort. Baseline demographic and pathophysiological variables were similar among the 3 groups except penile length and previous PD medications. Following treatment, group 3 demonstrated significantly greater improvements in curvature (mean, 20.3°/31% for group 1, 19.2°/30% for group 2, and 33.8°/49% for group 3), length (-0.7 cm/-4%, -0.4 cm/-2%, and +1.9 cm/+17%, respectively), and subjectively estimated curvature improvement (44%, 32%, and 63% respectively), despite shorter daily PTT use (0.9 vs 1.9 hours/day). Group 3 was more likely than the other groups to experience ≥20°, ≥20%, and ≥50% curvature improvements, ≥1 cm length gain, and ≥20% length improvement. All results were statistically significant for group 3 versus groups 1 and 2, but not between groups 1 and 2, even after controlling for baseline features and isolating a subset of ≥3 hours/day PTT use (group 2). Group 3 was 6.9 times more likely to achieve ≥20° curvature improvement, and 3.5 times more likely to achieve ≥50% curvature improvement, and 10.7 times more likely to experience ≥20% length improvement. Adverse events were similar among the 3 groups. CLINICAL IMPLICATIONS: Use of the RestoreX device enhances mean curvature outcomes by 71% and increases penile length in men with PD receiving CCH therapy. STRENGTHS & LIMITATIONS: Study strengths include a prospective registry, consistent assessments, the largest single-site series with complete posttreatment outcomes reported to date, the largest PTT series reported to date, and a true-to-life clinical design. Limitations include the nonrandomized methodology and single-site setting. CONCLUSION: The combination of RestoreX and CCH is associated with significantly greater curvature and length improvements compared with CCH alone or CCH with other PTT devices. Alom M, Sharma KL, Toussi A, et al. Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile Traction Therapy in Men with Peyronie's Disease. J Sex Med 2019;16:891-900.

Mitogen-induced distinct epialleles are phosphorylated at either H3S10 or H3S28, depending on H3K27 acetylation.

Stimulation of the MAPK pathway results in mitogen- and stress-activated protein kinase 1/2 (MSK1/2)-catalyzed phosphorylation of histone H3 at serine 10 or 28 and expression of immediate-early (IE) genes. In 10T1/2 mouse fibroblasts, phosphorylation of H3S10 and H3S28 occurs on different H3 molecules and in different nuclear regions. Similarly, we show that mitogen-induced H3S10 and H3S28 phosphorylation occurs in separate pools in human primary fibroblasts. High-resolution imaging studies on both cell types reveal that H3S10 and H3S28 phosphorylation events can be induced in a single cell but on different alleles, giving rise to H3S10ph and H3S28ph epialleles. Coimmunoprecipitation and inhibition studies demonstrate that CBP/p300-mediated H3K27 acetylation is required for MSK1/2 to phosphorylate S28. Although the K9ac and S10ph marks coexist on H3, S10 phosphorylation is not dependent on K9 acetylation by PCAF. We propose that random targeting of H3S10 or H3S28 results from the stochastic acetylation of H3 by CBP/p300 or PCAF, a process comparable to transcriptional bursting causing temporary allelic imbalance. In 10T1/2 cells expressing Jun, at least two of three alleles per cell were induced, a sign of high expression level. The redundant roles of H3S10ph and H3S28ph might enable rapid and efficient IE gene induction.

CYP17 polymorphism (rs743572) is associated with increased risk of gallbladder cancer in tobacco users.

Gallbladder cancer (GBC) involves interplay of sex steroids, including estrogen and progesterone. Since CYP17 is a key enzyme involved in estrogen and testosterone hormone biosynthesis as well as in xenobiotic metabolism, it may be a potential candidate gene in the carcinogenesis of the gallbladder. Hence, the present study aimed to investigate the association of CYP17 (rs2486758, and rs743572) polymorphisms with GBC susceptibility. The present study included a total of 414 histologically confirmed GBC and 230 healthy controls. The CYP17 (rs2486758 and rs743572) polymorphisms were genotyped by TaqMan-Allele discrimination assays. Statistical analysis was performed by using SPSS ver. 16. Overall, both the CYP17 SNPs did not indicate any association with GBC risk at genotype, haplotype, or at the genotypic interaction levels. However, in the case-only analysis, CYP rs743572 showed association with increased risk of GBC in tobacco users at hetero genotype and dominant models, as compared to non-user GBC patients. The TCrs2486758-AGrs743572 genotypic combination was also associated with increased GBC susceptibility in tobacco users. CYP17 rs743572 is associated with increased risk of GBC in tobacco users in the North Indian population. However, the study requires confirmation in other populations.

Death receptor (DR4) haplotypes are associated with increased susceptibility of gallbladder carcinoma in north Indian population.

BACKGROUND AND AIM: Defective apoptosis is a hallmark of cancer development and progression. Death receptors (DR4, FAS) and their ligands (TRAIL, FASL) are thought to mediate the major extrinsic apoptotic pathway in the cell. SNPs in these genes may lead to defective apoptosis. Hence, the present study aimed to investigate the association of functional SNPs of DR4 (rs20575, rs20576 and rs6557634), FAS (rs2234767) and FASL (rs763110) with gallbladder cancer (GBC) risk. METHODS: This case-control study included 400 GBC and 246 healthy controls (HC). Genotyping was carried out by Taqman genotyping assays. Statistical analysis was performed by using SPSS ver16. Meta-analysis was performed using Comprehensive Meta-analysis software (Version 2.0, BIOSTAT, Englewood, NJ) to systematically summarize the possible association of SNP with cancer risk. Functional prediction of these variants was carried out using Bioinformatics tools (FAST-SNP, F-SNP). False discovery rate (FDR test) was used in multiple comparisons. RESULTS: The DR4 C rs20575 A rs20576 A rs6557634, G rs20575 A rs20576 G rs6557634 and G rs20575 C rs20576 G rs6557634 haplotypes conferred two-fold increased risk for GBC. Among these, the DR4 C rs20575 A rs20576 A rs6557634 haplotype emerged as main factor influencing GBC susceptibility as the risk was not modulated by gender or gallstone stratification. Our meta-analysis results showed significant association of DR4 rs6557634 with overall cancer risk, GI cancers as well as in Caucasians. We didn't find any association of FAS and FASL SNPs with GBC susceptibility. CONCLUSIONS: The DR4 haplotype C rs20575 A rs20576 A rs6557634 represents an important factor accounting the patients susceptibility to GBC probably due to decreased apoptosis. However, additional well-designed studies with larger sample size focusing on different ethnicities are required to further validate the results.

Association of adrenergic receptor gene polymorphisms in gallbladder cancer susceptibility in a North Indian population.

PURPOSE: Gallbladder cancer (GBC), the most common gastrointestinal and biliary tract malignancy, often coincides with gallstone disease (GSD). The genetic variants of adrenergic receptor (ADR) have been previously reported to be associated with hypomotility disorder of cardiovascular system and GSD. Since GSD may function as GBC precursor, the present study aimed to investigate the association of common functional genetic variants of ADRA2A C-1291G, ADRß3 T190C or Trp64Arg, and ADRß1 C1165G or Arg389Gly with GBC and GSD susceptibility. METHODS: The present study included a total of 400 histologically confirmed GBC, 230 GSD, and 268 healthy controls. The ADRA2A C-1291G, ADRß3 T190C, and ADRß1 C1165G polymorphisms were determined by PCR-RFLP. Statistical analysis was performed by using SPSS version 16. RESULTS: ADRß3 T190C polymorphism was significantly associated with increased risk of GBC (CT: Pcorr = 0.015, OR 2.87; CC: Pcorr = 0.038, OR 10.33; C allele: Pcorr = 0.014, OR 2.7; CT + CC: Pcorr = 0.017, OR 3.05). These associations existed even after gallstone and gender stratification. Similarly, ADRß3 T190C polymorphism was also associated with GSD risk, though limited only to female GSD patients. In contrary, ADRA2A C-1291G conferred a marginally increased risk only in GSD patients. ADRß1 C1165G polymorphism was not associated with GBC and GSD susceptibility when compared to controls. CONCLUSION: ADRß3 T190C polymorphism is significantly associated with GBC and GSD susceptibility. The ADRß3 T190C may be involved in the pathophysiology of GBC by both gallstone-dependent pathway and by some other independent mechanisms.

PSCA gene variants (rs2294008 and rs2978974) confer increased susceptibility of gallbladder carcinoma in females.

BACKGROUND AND AIM: PSCA is a tissue specific tumor suppressor or oncogene which has been found to be associated with several human tumors including gallbladder cancer. It is considered to be involved in the cell-proliferation inhibition and/or cell-death induction activity. Therefore, we aimed to investigate the role of PSCA gene polymorphisms in gallbladder cancer risk in North Indian population. METHODOLOGY: A total of 405 gallbladder cancer patients and 247 healthy controls were included in the case-control study for risk prediction. We examined the association of two functional SNPs, rs2294008 and rs2978974 in PSCA gene by genotyping using Taqman allelic discrimination assays. Statistical analysis was done using SPSS software, version 17. Linkage disequilibrium and haplotype analysis was done with the help of SNPstats software. FDR test was used to correct for multiple comparisons. RESULTS: No significant associations of rs2294008 and rs2978974 genetic variants of the PSCA gene were found with GBC risk at allele, genotype or haplotype levels. Stratifying the subjects on the basis of gallstone also did not show any significant result. However, on gender stratification, we found a significant association of Trs2294008-Grs2978974 haplotype with higher risk of GBC in females (FDR Pcorr=0.021, OR=1.6). In contrary, Trs2294008-A rs2978974 haplotype conferred significant lower risk in males (FDR Pcorr=0.013; OR=0.25). CONCLUSIONS: These findings suggest that PSCA genetic variants may have a significant effect on GBC susceptibility in a gender specific manner.

DCC (deleted in colorectal carcinoma) gene variants confer increased susceptibility to gallbladder cancer (Ref. No.: Gene-D-12-01446).

BACKGROUND AND AIM: GBC is a lethal and multifaceted disease. Deleted in colorectal carcinoma (DCC) is a well known tumor suppressor gene. Recently a small genome-wide association study (GWAS) identified DCC to be significantly associated with gallbladder cancer (GBC) susceptibility in a Japanese population sample. However, the study sample size was small and lacked independent replication. Therefore, the present study was carried out to replicate the association of two GWAS identified DCC SNPs (A>Grs4078288, C>Trs7504990) and two other SNPs (C>Grs2229080 and A>Grs714) previously associated with various cancers. METHODOLOGY: The study was accomplished in 406 GBC cases and 260 healthy control samples from North India. Genotyping was carried out by PCR-RFLP and Taqman genotyping assays. Statistical analysis was performed by using SPSS ver16 and functional prediction of these variants was carried out using Bioinformatics tools (FAST-SNP, F-SNP). RESULT: We did not observe association with GWAS-identified SNPs of DCC but other SNPs showed significant association. In addition, a DCC haplotype Grs2229080-Ars4078288-Crs7504990-Ars714 conferred high risk of GBC in India. The haplotype associated risk was independent of gallstone, sex or tobacco usages which are well-known modifiers of GBC risk. Further analysis suggested DCC A>Grs714 as a major risk conferring SNP in the Indian population. CONCLUSION: This study re-affirms the role of plausible tumor suppressor DCC variants, in gallbladder carcinogenesis and the risk haplotype may be explored as a useful marker for GBC susceptibility.

Higher risk of matrix metalloproteinase (MMP-2, 7, 9) and tissue inhibitor of metalloproteinase (TIMP-2) genetic variants to gallbladder cancer.

BACKGROUND: Matrix metalloproteinase belong to family of pericellular collagenases which degrade extracellular matrix (ECM), and is involved in the modulation and susceptibility of various cancers. METHODOLOGY: The present study included 410 gallbladder (GBC) cases and 230 healthy controls from North India. Study examined the associations of polymorphisms of MMP-2c.735C>T (rs2285053), MMP-2c.1306 C>T (rs243865), MMP7c.181A>G (rs11568818), MMP-9p.R279Q (rs17556) MMP-9p.P574R (rs2250889), MMP-9 p.R668Q (rs17577) and TIMP2c.418 G>C (rs8179090) to GBC susceptibility. Genotyping was carried out by PCR-RFLP. Statistical analysis was performed by using SPSS ver16. RESULTS: The MMP-2 c.735 [CT+TT], MMP-2c.1306 [CT+TT], MMP7 c.181 [AG+GG] and MMP-9 p.668 [RQ+QQ],TIMP2c.418 [GG+GC] genotypes were significantly associated with increased risk of GBC (P = 0.01; [OR]1.87, P = 0.02; [OR] 1.68, P = 0.02; [OR]=1.61, P = 0.002; [OR]=1.91,P = 0.01; [OR]=1.78 and (P = 0.03; [OR]=1.68; P = 0.01; [OR]=1.78 respectively). Haplotypes [C(-735) -T(-1306) ] and [T(-1306) -C(-735) ] of MMP-2 (P = <0.005; [OR] =1.78 P = <0.0001; [OR] =2.09) and haplotype [Q(279) -P(574) -Q(668) ]of the MMP-9 (P = 0.04; [OR] =2.75) were significantly associated with GBC risk. On stratification of GBC patients with/without gallstones, MMP-2 haplotypes were associated with higher GBC risk in patients accompanying gallstones whereas MMP-9 haplotypes showed risk in patients without stones. Combined effect of > 3 MMP/TIMP variant containing genotypes imparted increased risk of GBC (P < 0.0001; [OR] =3.36). Multivariate logistic regression results also supported association of MMP-2 (c.735C>T, c.1306 C>T), MMP-9 p.R668Q and TIMP2c.418G>C variants with GBC susceptibility. CONCLUSION: This study suggests that genetic variants in MMP-2,7,9 and TIMP-2genes are associated with higher susceptibility of gallbladder cancer.

Association of matrix metalloproteinase-7 (-181A>G) polymorphism with risk of esophageal squamous cell carcinoma in Kashmir Valley.

BACKGROUND/AIM: Degradation of the basement membrane and extracellular matrix by matrix metalloproteinases (MMPs) is believed to be an essential step in the complicated process of hematogenous metastasis. Matrix metalloproteinase-7 (MMP-7) is a small secreted proteolytic enzyme with a broad substrate specificity, and its expression has been shown to be associated with tumor invasion and metastasis for various cancers. PATIENTS AND METHODS: To document the role of MMP-7 polymorphism in esophageal carcinogenesis, a case-control study was performed comprising 135 patients with esophageal cancer (EC) and 195 healthy controls. Genotyping was done by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Data were statistically analyzed using χ2 - test and logistic regression models. RESULTS: Carriers for the MMP-7 (-181A>G) GG were associated with an increased risk for EC [odds ratio (OR = 2.17; 95% confidence interval (CI) = 1.21-3.92; P = 0.010; P-trend = 0.04]. Also, in a recessive model, our results showed that MMP-7 (-181A>G) GG allele conferred significantly higher risk for EC (OR =2.16; 95% CI = 1.31-3.54; P = 0.003). The high risk due to MMP-7 (-181GG) genotype was limited to squamous cell histology of EC (OR = 2.41; 95% CI = 1.27-4.56; P = 0.007). Although smoking (Hukka) and high consumption of salted tea are independent risk factors for EC, the interaction of MMP-7 (-181A>G) genotypes with these factors did not further modulate the risk of EC. CONCLUSIONS: In conclusion, our results show that MMP-7 (-181A>G) GG carriers are at a higher risk of esophageal squamous cell carcinoma in Kashmir valley.