Association of Lecithin Cholesterol Acyltransferase rs5923 Polymorphism in Iranian Individuals with Extremely Low High-Density Lipoprotein Cholesterol: Tehran Lipid and Glucose Study

Background:

The serum concentration of high-density lipoprotein cholesterol (HDL-C) is one of the important heritable risk factors for cardiovascular disease and is a target for therapeutic intervention. In this study, we aimed to evaluate the effects of lecithin cholesterol acyltransferase (LCAT) gene polymorphism rs5923 on LCAT enzyme activity and serum HDL-C concentration.

Methods:

The study population was selected from consecutive individuals with HDL-C ≤ 5^th percentile (n = 73) and extremely high HDL-C ≥ 95^th percentile (n = 57) who had participated in the Tehran Lipid and Glucose Study. The rs5923 polymorphism was genotyped using direct sequencing. LCAT activity was measured by fluorometric assay kit, and lipid concentrations were measured using the enzymatic colorimetric method.

Results:

The genotype frequencies were significantly different between the high HDL-C group (CC 94.7%, CT 5.3%) and the low HDL-C group (CC 83.6%, CT 16.4%) (P = 0.048). The T-allele frequencies in subjects with low and high HDL-C were 0.082 and 0.026, respectively (P = 0.16). The association of the single-nucleotide polymorphism rs5923 with low HDL-C was not statistically significant after adjustment for age, sex, and BMI (odd ratio = 2.65, 95% confidence interval = 0.32-21.5, P = 0.36, regression logistic analysis). Also, the effects of LCAT enzyme activity did not depend on the HDL-C level (P = 0.24).

Conclusion:

rs5923 polymorphism is not associated with low HDL-C levels in Iranian population. Key Words: Polymorphism, Single nucleotide, Lipoproteins     

Molecular Genetic Analysis of the Variable Number of Tandem-Repeat Alleles at the Phenylalanine Hydroxylase Gene in Iranian Azeri Turkish Population

Background:

The variable numbers of tandem-repeat (VNTR) alleles at the phenylalanine hydroxylase (PAH) gene have been used in carrier detection and prenatal diagnosis in phenylketonuria families. This study was carried out to analyze VNTR alleles at the PAH gene in Iranian Azeri Turkish population.

Methods:

In this study, 200 alleles from general population were studied by PCR.

Results:

The frequencies of VNTR alleles were 45%, 46%, 2%, 3%, 1%, and 3% in studied group regarding 3, 8, 9, 11, 12, and 13 repeat copies, respectively. Statistically significant differences were not found between expected and observed frequencies of VNTR genotypes (P > 0.05).

Conclusions:

VNTR alleles with three and eight repeats were frequent, and the VNTR alleles with 13 repeats showed 3% frequency in the tested group. This study is the first report on tested population genetic structure using VNTR alleles at the PAH gene. Key Words: Phenylalanine hydroxylase, Population genetics, Variable numbers of tandem-repeat     

A Novel Mutation in the Aprataxin (APTX) Gene in an Iranian Individual Suffering Early-Onset Ataxia with Oculomotor Apraxia Type 1(AOA1) Disease

Background

Ataxia with oculomotor apraxia type 1 (AOA1) shows early onset with autosomal recessive inheritance and is caused by a mutation in the aprataxin (APTX) gene encoding for the APTX protein.

Methods

In this study, a 7-year-old girl born of a first-cousin consanguineous marriage was described with early-onset progressive ataxia and AOA, with increased cholesterol concentration and decreased albumin concentration in serum. PCR and direct DNA sequencing was performed after DNA extraction.

Results

Sequencing analysis revealed a novel homozygous deletion in c.643 and A>T single nucleotide polymorphism in c.641 in exon 6 of the APTX gene [ENST00000379825].

Conclusion

It seems that this region of exon 6 is probably a hot spot; however, no deletions have been reported in exon 6 yet. Key Words: Ataxia oculomotor apraxia 1 (AOA1), aprataxin (APTX), Iranian     

Impact of Methylenetetrahydrofolate Reductase C677T Polymorphism on the Risk of Gastric Cancer and Its Interaction with Helicobacter pylori Infection

Background

Attempts for early detection of gastric cancer have recently focused on host''s genetic susceptibility factors and gene-environment interactions. We have, herein, studied the association of MTHFR C677T single nucleotide polymorphism (SNP) and its interaction with Helicobacter pylori infection, smoking, age and gender on the risk of gastric cancer among an Iranian population.

Methods

Gastric cancer patients (n = 450) and cancer-free controls (n = 780) were studied for serum H. pylori-specific IgG antibodies by ELISA and MTHFR C677T polymorphism (SNP) by PCR-RFLP. Demographic and life style data were collected through patient interviews. Unconditional logistic regression model estimated odds ratio (OR) and the corresponding 95% confidence intervals (CI).

Results

The interactions of MTHFR genotype with H. pylori infection (P = 0.03), age (P = 0.049) and gender (P = 0.007) were statistically significant. Accordingly, MTHFR C677T carriers who were also positive for H. pylori infection exhibited 80% (OR = 1.8, 95% CI = 1.0-2.9) significant excess risk of non-cardia gastric cancer. Furthermore, subjects over the age of 50 or female subjects carrying MTHFR C677T SNP showed 40 (OR = 1.4, 95% CI = 1.0-2.0) and 100 (OR = 2.0, 95% CI = 1.2-3.2) percent increased risk of gastric cancer, respectively.

Conclusion

MTHFR C677T SNP seems to increase the risk of gastric cancer and the effect is significantly inflated by interactions with H. pylori infection, age and gender. Key Words: Helicobacter pylori, Smoking, Gender identity, Age group, Methylenetetrahydrofolate reductase     

Association Study of rs1333040 and rs1004638 Polymorphisms in the 9p21 Locus with Coronary Artery Disease in Southwest of Iran

Background:

Coronary artery disease (CAD) is a multifactorial and heterogenic disease. Recently, genome-wide association studies have reported that rs1333040 (C/T) and rs1004638 (A/T) single nucleotide polymorphisms (SNPs) in the 9p21 locus have very strong association with CAD. This study aimed to examine these associations in Southwest of Iran.

Methods:

Blood samples were collected from 200 CAD patients and 110 healthy individuals with no CAD. The association of two SNPs with CAD was evaluated by PCR and restriction fragment length polymorphism.

Results:

Chi-square test showed no association between rs1333040 SNP and CAD (X²: 4.66, df: 2, P=0.09). Also, there was no association between rs1004638 SNP and CAD (X²: 0.27, df: 2, P=0.88).

Conclusion:

No association was observed between rs1333040 and rs1004638 SNPs in the 9P21 region and CAD in Southwest of Iran. Key Words: Coronary artery disease, Single nucleotide polymorphisms, Genetic association study, Iran     

Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ ² P > 0.05), including only one common haplotype (GenBank access no. {"type":"entrez-nucleotide","attrs":{"text":"EF413075","term_id":"151936076","term_text":"EF413075"}}EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran     

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression

Background:

Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors.

Methods:

In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48, or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student’s t-test, and P<0.05 was considered statistically significant.

Results:

Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05).

Conclusions:

Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.Key Words: Adiponectin, Adiponectin receptors, Endometriosis, Stromal cells     

Prevalence of Class 1 Integrons and Extended Spectrum Beta Lactamases among Multi-Drug Resistant Escherichia coli Isolates from North of Iran

Background:

Extended spectrum beta lactamases (ESBLs) are an important cause of transferable multidrug resistance (MDR) in gram-negative bacteria. The most described ESBL genes are generally found within integron-like structures as mobile genetic elements. The aim of this study was to identify the accompanying of class 1 integrons and ESBLs in the MDR E. coli isolates.

Methods:

Susceptibility to antimicrobial agents was determined for 33 E. coli strains by the disk diffusion method. Double-disk synergy test was applied for screening ESBL. To identify the strains carrying integrons, the conserved regions of integron-encoded integrase gene intI1 were amplified. For detection of gene cassettes, 5′CS and 3′CS primers were used.

Results:

All E. coli isolates were identified as multi-drug resistant. More than 50% of the isolates were resistant to tetracycline, cephalothin, cefuroxime, amoxicillin-clavulanic acid, and third generation cephalosporines. Nearly all of the isolates displayed sensitivity to piperacillin. There was a significant correlation between production of ESBL and resistance to all antibiotics except for ciprofloxacin and piperacillin (P < 0.01). Thirty two MDR strains (97%) included class 1 integron, and some isolates that included integrons were similar in the size of gene cassettes. The isolates were different in the resistance profiles; however, some others had similar resistance profiles. Of eight ESBL positive isolates, seven (87.5%) carried class 1 integrons.

Conclusion:

Class 1 integrons were frequent in MDR and also ESBL-producing E. coli isolates. High prevalence of class 1 integrons confirms that integron-mediated antimicrobial gene cassettes are important in E. coli resistance profile. Key Words: Antibiotic, Integrons, Escherichia coli     

Identification of Sequence Variation in the Apolipoprotein A2 Gene and Their Relationship with Serum High-Density Lipoprotein Cholesterol Levels

Background:

Apolipoprotein A2 (APOA2) is the second major apolipoprotein of the high-density lipoprotein cholesterol (HDL-C). The study aim was to identify APOA2 gene variation in individuals within two extreme tails of HDL-C levels and its relationship with HDL-C level.

Methods:

This cross-sectional survey was conducted on participants from Tehran Glucose and Lipid Study (TLGS) at Research Institute for Endocrine Sciences, Tehran, Iran from April 2012 to February 2013. In total, 79 individuals with extreme low HDL-C levels (≤5^th percentile for age and gender) and 63 individuals with extreme high HDL-C levels (≥95^th percentile for age and gender) were selected. Variants were identified using DNA amplification and direct sequencing.

Results:

Screen of all exons and the core promoter region of APOA2 gene identified nine single nucleotide substitutions and one microsatellite; five of which were known and four were new variants. Of these nine variants, two were common tag single nucleotide polymorphisms (SNPs) and seven were rare SNPs. Both exonic substitutions were missense mutations and caused an amino acid change. There was a significant association between the new missense mutation (variant Chr.1:16119226, Ala98Pro) and HDL-C level.

Conclusion:

None of two common tag SNPs of rs6413453 and rs5082 contributes to the HDL-C trait in Iranian population, but a new missense mutation in APOA2 in our population has a significant association with HDL-C.     

Protective Effect of Vitamin E against Diabetes-Induced Oxidized LDL and Aorta Cell Wall Proliferation in Rat

Background:

Hyperlipidemia and oxidized-low-density lipoproteins (Ox-LDL) are important independent cardiovascular risk factors that have been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. The purpose of the present study was to investigate the effect of vitamin E on Ox-LDL, lipid profile, C-reactive protein (CRP), and VSMC proliferation of rat aorta.

Methods:

Male Wistar rats (n = 32) were divided into four groups namely: sham (SH), control (C), non-treated diabetic, and vitamin E-treated diabetic (VETD) groups. Ox-LDL, lipid profile, CRP and VSMC proliferation of aorta were measured after 42 days.

Results:

The results revealed that along with a significant increase in VSMC proliferation, the amount of CRP, Ox-LDL, and lipid profiles in diabetic rats. VSMC proliferation was significantly ameliorated, and elevated CRP, Ox-LDL, and lipid profiles were also restored to those of shams in VETD.

Conclusions:

These findings strongly support the idea that diabetes induces Ox-LDL-mediated oxidative stress and VSMC proliferation in aorta of rat and imply that vitamin E has a strong protective effect as an antioxidant. Key Words: Ox-LDL, Vitamin E, Diabetes, VSMC proliferation     

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

Expression Analysis of mir-21 and mir-221 in Cancerous Tissues from Iranian Patients with Gastric Cancer

Background:

Early detection is a key to survival for gastric cancer. Molecular markers such as miRNA (microRNA) can have great importance in the early diagnosis of gastric cancer. Expression of miR-21 and miR-221 are deregulated in many types of human cancers. This study aimed to investigate the differences in miRNA expression patterns within the Iranian population.

Methods:

Total RNA was extracted from gastric cancer tissues and adjacent non-cancerous tissues from 32 patients. Expression levels of miR-21 and miR-221 were detected by Real time RT-PCR using a specific primer, with 5s rRNA as the internal reference gene.

Results:

Our data showed that the expression levels of miR-21 and miR-221 in gastric cancer samples were significantly higher than in paired non-cancerous samples (P < 0.05). The receiver operating characteristic (ROC) analyses yielded the area under the curve (AUC) values of 80.30 for miR-21 and 93.30 for miR-221, and combined ROC analysis revealed the highest AUC value of 96.90 in discriminating GC patients from healthy controls.

Conclusion:

It seems that miR-21 and miR-221 expression pattern in Iranian patients with gastric cancer are similar to any other population. Considering the increased expression level of two miRNAs in cancerous tissue compared to normal tissue as well as the area under ROC curve, miR-21 and miR-221 can be used for early detection of gastric cancer. Key Words: MicroRNAs, Tumor markers, Stomach neoplasms     

Nomenclature report on rice WRKY's - Conflict regarding gene names and its solution

Background

Since whole genome sequences of rice were made publically accessible, the number of articles on new rice genes has increased remarkably. The Committee on Gene Symbolization, Nomenclature and Linkage (CGSNL) of the Rice Genetics Cooperative published the gene nomenclature system for rice and encouraged researchers to follow the rules before publishing their results. The CGSNL provides an on-line registration system for newly identified rice genes to prevent conflicts and/or duplication of gene name in journal articles.

Findings

Recently, the CGSNL surveyed genes in the rice WRKY family in published journal articles and found several duplicated gene names.

Conclusions

To discuss and resolve inconsistencies in WRKY gene nomenclature, the rice WRKY working group was established and redefined the nomenclature. This report announces the conclusion.     

Protective Effects of Restricted Diet and Antioxidants on Testis Tissue in Rats Fed with High-Fat Diet

Background:

A high-fat diet (HFD) promotes the oxidative stress formation, which in turn has hazardous effects on reproductive system and fertility. The present study examines the potential positive effects of a restricted high-fat diet (RHFD) and antioxidants consumption on sperm parameters and testis tissue in rats.

Methods:

Male rats (n = 48) were divided into four groups (12 in each group): control group (Cont), HFD group, RHFD, and RHFD with astaxanthin and vitamins E and C group (RHFDA). After 12 weeks, serum analysis and sperm parameters study were performed. Sections of fixed testes were stained with Hematoxilin and Eosin to study the histological changes. A one-way ANOVA was used to compare the data.

Results:

HFD fed animals presented significant increase in weight load and serum low density lipoprotein (LDL-C) levels (P < 0.05). The sperm count in RHFD was lower than three other groups (P < 0.05) and sperm motility of RHFDA group was significantly higher than HFD and RHFD groups (P < 0.05). The histological study was showed a significant increase in spermatogonium number in RHFDA compared to three other groups (P < 0.05). The number of spermatocyte I and spermatid in RHFD was significantly (P < 0.05) lower than Cont and HFD groups.

Conclusion:

HFD and obesity can affect sperm parameters and spermatogenesis and antioxidants consumption may improve their quality. Although the RHFD is a benefit way in weight loss and decrease of LDL-C of serum, but it is suggested that is not effective on sperm quality improvement.Key Words: Spermatogenesis, high-fat Diet, Restricted fat diet, Antioxidants, Astaxanthin     

Bacteria Etiological Agents Causing Lower Respiratory Tract Infections and Their Resistance Patterns

Background:

Lower respiratory tract infections (LTRIs) are among the most common infectious diseases with potential life-threatening complications.

Methods:

The study consisted of 426 patients with suspected LTRIs from mid and far western region of Nepal between September 2011 and July 2014. The specimens were collected and processed according to the standard microbiological methods at the Central Laboratory of Microbiology of Nepalgunj Medical College, Nepal.

Results:

Among the isolated Gram-positive organisms, Streptococcus pneumonia (n = 30, 51.7%) was the most predominant pathogen, followed by Staphylococcus aureus (n = 28, 48.3%). Among the isolated Gram-negative organisms, Pseudomonas aeruginosa (n = 71, 35.32%) was the most predominant pathogen, followed by Haemophilus influenzae (n = 68, 33.83%), Klebsiella pneumonia (n = 36, 17.19%), and Escherichia coli (n = 26, 12.94%). The pattern of resistance varied regarding the bacteria species, and there were multi-resistant isolates. Also, a significant difference (P < 0.05) was observed between males and females for each type of bacterial species. Among 259 isolates, 86 (33.20%) were from children aged 1-10 years, which were statistically significant (P < 0.05) compared to the other age groups.

Conclusions:

P. aeruginosa and H. influenzae (Gram-negative) and S. pnemoniae (Gram-positive) were the most common bacterial isolates recovered from LTRIs. Age group of 1-10 years old was at a higher risk. Many isolates showed appreciable levels of antibiotic resistance due to antibiotic abuse. There is a need to increase surveillance and develop better strategies to curb the increasing prevalence of LRTI in this region of Nepal.Key Words: Bacterial infections, Antimicrobial drug resistance, Respiratory system, Nepal     

Particular Distribution of Enterobacter cloacae Strains Isolated from Urinary Tract Infection within Clonal Complexes

Background:

Based on biochemical properties, Enterobacter cloacae represents a large complex of at least 13 variant species, subspecies, and genotypes that progressively identified as the most species causing hospital-acquired infections. The aim of this study was to determine the relevance between phylogenetically related strains within the E. cloacae complex and the frequency of urinary tract infection caused by them.

Methods:

A 268-bp fragment was obtained from hsp60 gene for 50 clinical E. cloacae isolates from urine cultures of inpatients that admitted to six hospitals in Tehran, Iran during December 2012 to November 2013. The 107 nucleotide sequences were analyzed and the evolutionary distances of sequences were computed and neighbor-joining tree was calculated.

Results:

It showed that all of the genetic clusters have not an equal involvement in pathogenesis of urinary tract infections. Three superior clusters were found, together representing more than two third (80%) of the isolates (cluster VI with 25 members; clusters III and VIII with 9 and 6 members, respectively) and some genetic clusters were absent (IV, X, XII, and xiii), some of which are supposed to be associated with plants and no human infection has been reported.

Conclusions:

This study, for the first time, reports the unequal contribution of E. cloacae complex subspecies and clusters in urinary tract infections in Iran and together with studies from other countries suggest that the subspecies of E.hormaechei subsp. Oharae is the most prevalent E. cloacae complex subspecies regardless of country under study. Key Words: Enterobacter cloacae complex, Urinary tract infection, hsp60     

Effect of Human Ovarian Tissue Vitrification/Warming on the Expression of Genes Related to Folliculogenesis

Background:

Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha (FIGLA), growth differentiation factor-9 (GDF-9), follicle-stimulating hormone receptor (FSHR), and KIT LIGAND after vitrification/warming of human ovarian tissue.

Methods:

Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group (control). Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF-9,, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups.

Results:

Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups (P > 0.05).

Conclusion:

Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes. Key Words: Vitrification, Gene expression, Humans