Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11β-hydroxylase deficiency

Context

The deficiency of steroid 11β-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension.

Objective

The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia.

Patient

The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years.

Results

The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X).

Conclusion

We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.     

Persistence length of fascin-cross-linked actin filament bundles in solution and the in vitro motility assay

Background

Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles.

Methods

Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: < cos(θ(0) − θ(s)) > = exp(−s / (2Lp)) where θ(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments.

Results

Bundle-Lp (bundles < 15 μm long) increased from ~ 10 to 150 μm with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 μm), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis.

Conclusions

Fascin–actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle.

General significance

The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin–actin bundles.     

CYP2C9 genotype and association with bone mineral density: A pilot study

Background

In recent years reduced bone mineral density (BMD) and osteoporosis have become major public health problems. Single nucleotide polymorphisms (SNPs) in the cytochrome P450 2C9 (CYP2C9) gene influence the response to oral anticoagulant drugs, which are positively associated with the risk to develop osteoporosis. The aim of the present investigation was to clarify a potential role of CYP2C9 sequence variations and susceptibility to develop osteoporosis.

Subjects and methods

Ninety two consecutive angiologic outpatients, mean age: 60.3 ± 14.4, without secondary causes of bone loss were genotyped and classified as patients with normal BMD, osteopenia and osteoporosis according to WHO criteria by dual-energy X-ray absorptiometry at the lumbar spine and/or the femoral neck. Potential association between the CYP2C9 genotype and BMD was tested.

Results

59% of the patients (n = 54) presented with reduced BMD and were compared to 38 age-matched persons with normal BMD. The genotype distribution showed 15% heterozygous for CYP2C9*2 p.Arg144Cys, 14% for CYP2C9*3 p.IIe359Leu, 2% for both polymorphisms, and 69% had wildtype genotypes. Patients with CYP2C9 mutations had significantly lower BMD values at the femoral neck and displayed a four-fold higher adjusted risk to suffer from reduced BMD than individuals with wildtype genotypes (p = 0.02).

Discussion

Oral anticoagulant treatment is common in angiologic outpatients. The gene variants CYP2C9*2 and CYP2C9*3 have been shown to require lower maintenance doses of oral anticoagulant drugs. An association between oral anticoagulant drugs and the susceptibility to develop osteoporosis in relation to sequence variations in the CYP2C9 gene is suggested to be mediated via the glucocorticoid synthesis pathway.

Conclusion

The CYP2C9*2/CYP2C9*3 variants were significantly associated with femoral BMD in a selected elderly Austrian population. These variants could contribute to the complex risk to develop osteoporosis.     

The contribution of platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms to platelet response in patients treated with aspirin

Objective

Aspirin is an antiplatelet agent commonly used in treatment of patients with high risk to develop stroke and myocardial infarction. However, inter-individual variability regarding the inhibition of platelet function by aspirin is well documented. In this study, the correlation between platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms and antiplatelet response in patients treated with aspirin was investigated.

Methods

Jordanian adult patients (n = 584) who are taking aspirin as an antiplatelet agent participated in the study. Platelet aggregation response was measured using Multiplate Analyzer® system. Polymerase chain reaction–restriction fragment length polymorphism assay (PCR–RFLP) was used for genotyping of the examined polymorphisms.

Results

Aspirin resistance was found in 15.8% of patients. Response to aspirin was significantly associated with GPIba C-5T polymorphism (P < 0.05). However, the GPIa C807T and COX-2G-765C polymorphisms were not related to aspirin resistance (P > 0.05).

Conclusion

A considerable fraction of the Jordanian population is resistant to the antiplatelet effect of aspirin, which might be related to GPIba C-5T polymorphism.     

CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease

Background

There are nearly 2000 cystic fibrosis transmembrane regulator (CFTR) mutations that cause cystic fibrosis (CF). These mutations are classified into six classes; on the one hand, the first three classes cause severe disease involvement in early childhood, on the other hand, the Class IV, V and VI mutations cause minor severe disease in the same age. Nowadays, with therapeutic advances in CF management and competence of pediatricians, physicians of adults have to deal with two groups of CF patients: (i) adults diagnosed in childhood with severe mutations and (ii) adults who initiated symptoms in adulthood and with Class IV, V and VI mutations. The aim of this study was to analyze adults from a clinical center, treated as CF disease, screening the CFTR genotype and evaluating the clinical characteristics.

Methods

Thirty patients followed as CF disease at the University Hospital were enrolled. After a complete molecular CFTR negative screening and sweat test levels between 40 and 59 mEq/L, five patients were characterized as non-CF disease and were excluded. Molecular screening was performed by CFTR gene sequencing/MLPA or by specific mutation screening. Clinical data was obtained from medical records. The patients were divided into three groups: (1) patients with Class I, II and III mutations in two CFTR alleles; (2) genotype with at least one allele of Class IV, V or VI CFTR mutations and, (3) non-identified CFTR mutation + one patient with one allele with CFTR mutation screened (Class I).

Results

There was an association of CFTR class mutation and sodium/chloride concentration in the sweat test (sodium: p = 0.040; chloride: p = 0.016), onset of digestive symptoms (p = 0.012), lung function parameter (SpO₂ — p = 0.016), Bhalla score (p = 0.021), age at diagnosis (p = 0.008) and CF-related diabetes (p = 0.029). There was an association between Pseudomonas aeruginosa chronic colonization (as clinical marker for the lung disease status) and lung impairment (FEV₁% — p = 0.027; Bhalla score — p = 0.021), CF-related diabetes (p = 0.040), chloride concentration in the sweat test (p = 0.040) and chronic infection by microorganisms (Staphylococcus aureus — p = 0.039; mucoid P. aeruginosa — p = 0.001). There is no positive association with the status of other clinical markers and the CFTR genotype groups. For clinical association with pancreatic insufficiency (as clinical marker for digestive symptoms), no association was related.

Conclusion

The adults with CF diagnosed by sweat test have specific clinical and genotypic characteristics, being a population that should be studied to cause better future management. Some patients treated as CF disease by clinical symptoms, showed no disease, taking into account the sweat test and complete exon sequencing/MLPA screening.     

A patient with partial trisomy 21 and 7q deletion expresses mild Down syndrome phenotype

Backround

Down syndrome (DS) is the most common aneuploidy in live-born individuals and it is well recognized with various phenotypic expressions. Although an extra chromosome 21 is the genetic cause for DS, specific phenotypic features may result from the duplication of smaller regions of the chromosome and more studies need to define genotypic and phenotypic correlations.

Case report

We report on a 26 year old male with partial trisomy 21 presenting mild clinical symptoms relative to DS including borderline intellectual disability. In particular, the face and the presence of hypotonia and keratoconus were suggestive for the DS although the condition remained unnoticed until his adult age array comparative genomic hybridization (aCGH) revealed a 10.1 Mb duplication in 21q22.13q22.3 and a small deletion of 2.2 Mb on chromosomal band 7q36 arising from a paternal translocation t(7;21). The 21q duplication encompasses the gene DYRK1.

Conclusion

Our data support the evidence of specific regions on distal 21q whose duplication results in phenotypes recalling the typical DS face. Although the duplication region contains DYRK1, which has previously been implicated in the causation of DS, our patient has a borderline IQ confirming that their duplication is not sufficient to cause the full DS phenotype.     

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.     

Role of CYP1A2*1F polymorphism in cancer risk: Evidence from a meta-analysis of 46 case–control studies

Background

Emerging evidence showed that the common polymorphism (CYP1A2*1F, rs762551 C → A) in the promoter region of the CYP1A2 gene might be associated with susceptibility to cancer in humans. But individually published results were inconclusive. The aim of this meta-analysis is to investigate the association between CYP1A2*1F polymorphism and cancer risk.

Methods

The Pubmed, Embase, Web of Science and Chinese BioMedical databases were searched for all articles published up to September 1st, 2012. Statistical analyses were performed using the STATA 12.0 software.

Results

Forty-six case–control studies were included with a total of 22,993 cancer cases and 28,420 healthy controls. Meta-analysis results showed that the A allele of CYP1A2*1F polymorphism was associated with a decreased cancer risk (odds ratio [OR] = 0.92, 95% confidence interval [CI]: 0.87–0.98, P = 0.013). In the subgroup analysis by cancer types, the A allele of CYP1A2*1F polymorphism may increase the risk of breast cancer (OR = 1.05, 95% CI: 1.01–1.10, P = 0.024), and is also associated with a decreased risk of ovarian cancer (OR = 0.70, 95% CI: 0.54–0.89, P = 0.004). However, similar results were not found in lung, colorectal, bladder, endometrial, pancreatic and gastric cancers. Further subgroup analysis by ethnicity also showed a significant association between the A allele of CYP1A2*1F polymorphism and a decreased cancer risk among Caucasian populations (OR = 0.91, 95% CI: 0.84–0.98, P = 0.014); but no significant associations were observed among Asian populations.

Conclusions

Results from the current meta-analysis indicate that the A allele of CYP1A2*1F polymorphism may be associated with breast and ovarian cancer risk, especially among Caucasian populations.     

Combined genetic mutations have remarkable effect on deep venous thrombosis and/or pulmonary embolism occurence

Purpose

Although deep vein thrombosis and thromboembolic diseases differ among various races, they are still important in our day. The difficulties in treatment and following-up of these diseases are caused by secret genetic mutations rather than predisposing factors.

Methods

Between January 2011 and May 2013, patients who were traced for deep vein thrombosis and/or pulmonary embolism were evaluated retrospectively. 84 patients (53.6% males and 46.4% females) were included in the study. Their family histories, predisposing factors and treatments were researched. Factor V Leiden (G 1691A), Factor II G20210A, Plasminogen Activator Inhibitor-Type 1 (4G/5G), and Methylene Tetrahydrofolate Reductase (C677T, A1298C) mutations were investigated from peripheral venous blood.

Results

Among the genetic mutations we searched, the incidence of single mutation rate was observed at 11.9%, double mutation collocation at 44%, triple mutation collocation at 29.8%, quadruple mutation collocation at 13.1%, and finally, quintuplet mutation collocation at 1.2%. Our approximate mutation number was found as 2.47 ± 0.91.

Conclusion

We observed that multiple mutations were high in number compared to single genetic mutations. The patients who have multiple mutations should be more in the front line considering their diagnosis, treatment and following up, and also in terms of decreasing mortality, morbidity and recurrence.     

XRCC3 Thr241Met polymorphism and risk of acute myeloid leukemia in a Romanian population

Background

DNA repair systems have a critical role in maintaining the genome integrity and stability. DNA repair gene polymorphisms may influence the capacity to repair DNA damage, and thus lead to an increased cancer susceptibility. X-ray repair cross-complementing groups 3 (XRCC3), a DNA repair gene, may be involved in acute myeloid leukemia susceptibility. The objective of the current study was to investigate the association of Thr241Met polymorphism of XRCC3 gene with the risk of acute myeloid leukemia (AML).

Methods

This study included 78 AML patients and 121 healthy individuals without cancer. We used polymerase chain reaction-restriction fragment length polymorphism assay to determine XRCC3 genotypes.

Results

The XRCC3 variant genotype (Thr/Met+Met/Met) was more frequent in AML patients than in healthy controls (OR = 2.76, 95% CI: 1.52-4.98, P = 0.001). Our study revealed a statistically significant association between variant genotype (Thr/Met+Met/Met) and AML de novo compared to secondary AML (P = 0.007). No significant associations were found between any genotype and age at diagnosis, number of white blood cells and subtype of AML. Overall survival of patients with Thr/Thr genotype was better than those of variant Thr/Met and Met/Met genotypes.

Conclusions

Our findings indicate that the XRCC3 Thr241Met polymorphism may be a genetic risk factor for AML, particularly in male patients with de novo AML from the central part of Romania.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Background

Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.

Methods

To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).

Results

DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.

Conclusions

DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.

General significance

The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.     

Variants in endothelial nitric oxide synthase (eNOS) gene in idiopathic infertile Brazilian men

Purpose

In recent years, considerable concern has been expressed about the deleterious effects of reactive oxygen species (ROS) on sperm function, because ROS at high levels is potentially detrimental to sperm function and quality. Nitric oxide (NO) is a powerful anti-oxidant present in seminal plasma. The aim of the study was to analyze the distribution of the of endothelial nitric oxide synthase (eNOS) gene (T-786C, G894T, e 4a/b) polymorphisms in idiopathic infertile Brazilian men and evaluate the possible role of these polymorphisms in sperm count.

Methods

A case–control study was performed comprising 208 infertile men [n = 74 with non-obstructive azoospermia and n = 134 with severe oligozoospermia] and 201 fertile men as controls. Genotyping of eNOS polymorphisms was performed by real time (T-786C and G894T) and conventional PCR (4a/b). The results were analyzed statistically and a p-value < 0.05 was considered significant.

Results

According to the sperm count, relatively similar eNOS polymorphism genotypes and allele frequencies were found among the groups. Combined genotypes of the eNOS polymorphisms did not identify a haplotype associated with idiopathic infertility, even when the patients were separated in non-obstructive azoospermia or severe oligozoospermia.

Conclusion

In conclusion, the findings demonstrate that, in Brazilian population studied, genetic variations, T-786C, G894T, and e 4a/b, of the eNOS gene are not associated with male infertility.     

Insight into genetic determinants of resting heart rate

Background

Recent studies suggested that resting heart rate (RHR) might be an independent predictor of cardiovascular mortality and morbidity. Nonetheless, the interrelation between RHR and cardiovascular diseases is not clear. In order to resolve this puzzle, the importance of genetic determinants of RHR has been recently suggested, but it needs to be further investigated.

Objective

The aim of this study was to estimate the contribution of common genetic variations on RHR using Genome Wide Association Study.

Methods

We performed a Genome Wide Association Study in an isolated population cohort of 1737 individuals, the Italian Network on Genetic Isolates — Friuli Venezia Giulia (INGI-FVG). Moreover, a haplotype analysis was performed. A regression tree analysis was run to highlight the effect of each haplotype combination on the phenotype.

Results

A significant level of association (p < 5 × 10^− 8) was detected for Single Nucleotide Polymorphisms (SNPs) in two genes expressed in the heart: MAML1 and CANX. Founding that the three different variants of the haplotype, which encompass both genes, yielded a phenotypic correlation. Indeed, a haplotype in homozygosity is significantly associated with the lower quartile of RHR (RHR ≤ 58 bpm). Moreover no significant association was found between cardiovascular risk factors and the different haplotype combinations.

Conclusion

Mastermind-like 1 and Calnexin were found to be associated with RHR. We demonstrated a relation between a haplotype and the lower quartile of RHR in our populations. Our findings highlight that genetic determinants of RHR may be implicated in determining cardiovascular diseases and could allow a better risk stratification.     

The impact of GGH -401C > T polymorphism on cisplatin-based chemoradiotherapy response and survival in cervical cancer

Aims

Cervical cancer is the third most frequent cancer in women worldwide, mostly treated with cisplatin-based chemoradiotherapy. Since it is known that folate metabolism might interfere with cisplatin effectiveness, we intended to study the influence of the Gamma Glutamyl Hydrolase -401C > T polymorphism in treatment response in cervical cancer.

Methods

We retrospectively reviewed the clinical data of 167 patients with bulky cervical cancer submitted to cisplatin-based chemoradiotherapy. The genotypes of GGH -401C > T SNP were determined by real-time PCR and statistical analysis was performed by χ² test and survival analysis.

Results

The genotypes of GGH-401C > T were significantly associated with the response to platinum-based chemoradiotherapy. Treatment response was higher in patients carrying the CC genotype, who presented a significant increased chance of treatment response (survival time in months/genotype: 91 for CC Vs 72 for CT/TT; p = 0.035, log rank test). A Cox regression analysis accordingly showed that the presence of the T allele was significantly linked to a worse treatment response (HR = 3.036; CI 95% 1.032-8.934, p = 0.044).

Conclusions

The results of our study suggested the potential interest of GGH -401C > T as a predictive factor of the outcome of cervical carcinoma treated with cisplatin-based chemoradiotherapy.     

Trimethylamine N-oxide suppresses the activity of the actomyosin motor

Background

During actomyosin interactions, the transduction of energy from ATP hydrolysis to motility seems to occur with the modulation of hydration. Trimethylamine N-oxide (TMAO) perturbs the surface of proteins by altering hydrogen bonding in a manner opposite to that of urea. Hence, we focus on the effects of TMAO on the motility and ATPase activation of actomyosin complexes.

Methods

Actin and heavy meromyosin (HMM) were prepared from rabbit skeletal muscle. Structural changes in HMM were detected using fluorescence and circular dichroism spectroscopy. The sliding velocity of rhodamine-phalloidin-bound actin filaments on HMM was measured using an in vitro motility assay. ATPase activity was measured using a malachite green method.

Results

Although TMAO, unlike urea, stabilized the HMM structure, both the sliding velocity and ATPase activity of acto-HMM were considerably decreased with increasing TMAO concentrations from 0–1.0 M. Whereas urea-induced decreases in the structural stability of HMM were recovered by TMAO, TMAO further decreased the urea-induced decrease in ATPase activation. Urea and TMAO were found to have counteractive effects on motility at concentrations of 0.6 M and 0.2 M, respectively.

Conclusions

The excessive stabilization of the HMM structure by TMAO may suppress its activities; however, the counteractive effects of urea and TMAO on actomyosin motor activity is distinct from their effects on HMM stability.

General significance

The present results provide insight into not only the water-related properties of proteins, but also the physiological significance of TMAO and urea osmolytes in the muscular proteins of water-stressed animals.     

Monogalactosyl diacylglycerol,a replicative DNA polymerase inhibitor,from spinach enhances the anti-cell proliferation effect of gemcitabine in human pancreatic cancer cells

Background

Gemcitabine (GEM) is used to treat various carcinomas and represents an advance in pancreatic cancer treatment. In the screening for DNA polymerase (pol) inhibitors, a glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), was isolated from spinach.

Methods

Phosphorylated GEM derivatives were chemically synthesized. In vitro pol assay was performed according to our established methods. Cell viability was measured using MTT assay.

Results

Phosphorylated GEMs inhibition of mammalian pol activities assessed, with the order of their effect ranked as: GEM-5′-triphosphate (GEM-TP) > GEM-5′-diphosphate > GEM-5′-monophosphate > GEM. GEM suppressed growth in the human pancreatic cancer cell lines BxPC-3, MIAPaCa2 and PANC-1 although phosphorylated GEMs showed no effect. MGDG suppressed growth in these cell lines based on its selective inhibition of replicative pol species. Kinetic analysis showed that GEM-TP was a competitive inhibitor of pol α activity with nucleotide substrates, and MGDG was a noncompetitive inhibitor with nucleotide substrates. GEM combined with MGDG treatments revealed synergistic effects on the inhibition of DNA replicative pols α and γ activities compared with GEM or MGDG alone. In cell growth suppression by GEM, pre-addition of MGDG significantly enhanced cell proliferation suppression, and the combination of these compounds was found to induce apoptosis. In contrast, GEM-treated cells followed by MGDG addition did not influence cell growth.

Conclusions

GEM/MGDG enhanced the growth suppression of cells based on the inhibition of pol activities.

General significance

Spinach MGDG has great potential for development as an anticancer food compound and could be an effective clinical anticancer chemotherapy in combination with GEM.     

Interleukin-23 receptor genetic variants contribute to susceptibility of multiple cancers

Aim

Interleukin-23 (IL-23) and IL-23 receptor (IL23R) play an important role during the T-helper 17 (Th17) cell-mediated inflammatory process as well as pathogenesis of multiple cancers. Several IL-23R single nucleotide polymorphisms (SNPs), especially rs6682925, rs10889677 and rs1884444 polymorphisms, are considered to have significant impacts on susceptibility of multiple cancers. A number of case-control studies have explored the role these genetic polymorphisms in development of carcinogenesis, but the conclusions are inconsistent. Therefore, we conducted this meta-analysis to systematically investigate the associations between the three genetic variants and multiple cancer risk.

Methods

A total of ten studies are eligible (12,211 patients and 14,650 controls). Pooled odds ratios (ORs) and the 95% confidence interval (95% CI) were appropriately calculated using either fixed-effect model or random-effect model.

Results

Significant associations between rs6682925 or rs10889677 polymorphism and cancer risk were found (OR = 1.11, 95% CI = 1.03–1.21, P = 0.007; or OR = 0.85, 95% CI = 0.71–0.92, P = 0.001). However, there was no such association between rs1884444 genotypes and cancer susceptibility (P > 0.05).

Conclusion

These findings reveal that the IL-23R rs6682925 and rs10889677 genetic variants play a more important part in pathogenesis of multiple cancers.     

SLC45A2 mutation frequency in Oculocutaneous Albinism Italian patients doesn't differ from other European studies

Background

Oculocutaneous Albinism (OCA) is a heterogeneous group of inherited diseases involving hair, skin and eyes. To date, six forms are recognized on the effects of different melanogenesis genes.OCA4 is caused by mutations in SLC45A2 showing a heterogeneous phenotype ranging from white hair, blue irides and nystagmus to brown/black hair, brown irides and no nystagmus. The high clinic variety often leads to misdiagnosis.Our aim is to contribute to OCA4 diagnosis defining SLC45A2 genetic variants in Italian patients with OCA without any TYR, OCA2 and TYRP1 gene defects.

Materials and methods

After the clinical diagnosis of OCA, all patients received genetic counseling and genetic test. Automatic sequencing of TYR, OCA2, and TYRP1 genes was performed on DNA of 117 albino patients. Multiplex Ligation-dependent Probe Amplification (MLPA) was carried out on TYR and OCA2 genes to increase the mutation rate. SLC45A2 gene sequencing was then executed in the patients with a single mutation in one of the TYR, OCA2, TYRP1 genes and in the patients, which resulted negative at the screening of these genes.

Results

SLC45A2 gene analysis was performed in 41 patients and gene alterations were found in 5 patients. Four previously reported SLC45A2 mutations were found: p.G100S, p.W202C, p.A511E and c.986delC, and three novel variants were identified: p.M265L, p.H94D, and c.1156+1G>A. All the alterations have been detected in the group of patients without mutations in the other OCA genes.

Conclusions

Three new variants were identified in OCA4 gene; the analysis allowed the classification of a patient previously misdiagnosed as OA1 because of skin and hair pigmentation presence. The molecular defects in SLC45A2 gene represent the 3.4% in this cohort of Italian patients, similar to other Caucasian populations; our data differ from those previously published by an Italian researcher group, obtained on a smaller cohort of patients.     

Interaction between gene A-positive Helicobacter pylori and human leukocyte antigen II alleles increase the risk of Graves disease in Chinese Han population: An association study

Objective

The current study explored the correlation of Helicobacter pylori and the polymorphisms of human leukocyte antigen II (HLA-II) alleles with Graves disease (GD).

Methods

A total of 216 patients with GD were recruited. 102 healthy volunteers constituted the control group. Levels of H. pylori immunoglobulin G (IgG) antibodies and H. pylori cytotoxin-associated gene A (CagA) IgG antibodies were detected using enzyme-linked immunosorbent assays. Molecular typing of the HLA-II alleles was conducted using polymerase chain reaction with sequence specific primers.

Results

H. pylori, particularly CagA-positive strains, HLA-DQA1*0201, and HLA-DQA1*0501 were associated with GD (P = 0.015, OR = 1.811; P = 0.000, OR = 3.085; P = 0.000, OR = 0.315; and P = 0.004, OR = 2.844, respectively). Patients with CagA-positive H. pylori and negative HLA-DQA1*0201 or positive HLA-DQA1*0501 were more likely exposed to GD compared with those with only one of these indices.

Conclusion

CagA-positive H. pylori, negative HLA-DQA1*0201, or positive HLA-DQA1*0501 may increase the risk of GD.