Polymerase Chain reaction-RFLP Research Articles

The evaluation of IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms in Egyptian patients with alopecia areata: a case–control study

BackgroundAlopecia areata (AA) is a non-scarring hair loss condition that usually affects the scalp. The exact pathogenesis is poorly understood; however, multiple factors like genetics, environmental, psychological, and immunological factors may have a role. The purpose of this study was to look into possible links between the functional interleukin-4 (IL-4) gene intron 3 variable number of tandem repeats (VNTR) and TNF-(rs1799964) gene polymorphism and AA susceptibility. This case–control study consisted of 79 unrelated patients and 156 age- and sex-matched healthy individuals as a control group. The Severity of Alopecia Tool was used to assess the extent of hair loss from the scalp. Polymerase chain reaction (PCR) with specific primers was used to determine IL-4 gene 70-bp VNTR polymorphism while polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) was used to investigate TNF-α (rs1799964) gene polymorphism.ResultsNone of the selected polymorphisms for both genotypes and alleles had statistical significance when patients and controls were compared with each other (p-values for IL-4 VNTR were 0.11, 0.74, 0.052 and 0.27 and for TNF-α polymorphism was 0.71, 0.43, 0.65 and 0.55, respectively, for codominant, dominant, recessive and overdominant models of inheritance, respectively). Furthermore, the same results were retrieved when the genotypes were compared with the patient’s clinical and demographic data (p-value > 0.05).ConclusionThe findings indicate that IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms are not linked to the development of AA in the Egyptian population.

Polymorphism at codon 31 of CDKN1A (p21) as a predictive factor for bevacizumab therapy in glioblastoma multiforme

Glioblastoma (GBM), a prevalent and malignant brain tumor, poses a challenge in surgical resection due to its invasive nature within the brain parenchyma. CDKN1A (p21, Waf-1), a cyclin-dependent kinase inhibitor, plays a pivotal role in regulating cell growth arrest, terminal differentiation, and apoptosis. The existence of natural variants of CDKN1A has been associated with specific cancer types. In this retrospective study, our objective was to identify polymorphic variants of CDKN1A, specifically c.93C > A (codon 31 Ser31Arg), and investigate its potential impact within the scope of bevacizumab therapy for glioblastoma multiforme. This study involved a cohort of 139 unrelated adult Chinese GBM patients in Taiwan. Genomic DNA extracted from tumor samples was utilized for genotyping using the polymerase chain reaction (PCR) restriction fragment length polymorphism method (PCR–RFLP analysis). Through unconditional logistic regression analysis, odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. Our findings unveiled that among these GBM patients, the distribution of codon 31 polymorphisms was as follows: 23.02% were Serine homozygotes (Ser/Ser), 27.34% were Arginine homozygotes (Arg/Arg), and 49.64% were Serine/Arginine heterozygotes (Ser/Arg). While CDKN1A c.93C > A polymorphisms did not exhibit a direct association with overall survival in GBM patients, noteworthy survival benefits emerged among individuals with Arg/Arg and Arg/Ser genotypes who received combined concurrent chemoradiotherapy (CCRT) and bevacizumab treatment compared to those who underwent CCRT alone. Our findings indicate a significant involvement of the CDKN1A c.93C > A polymorphism in the development and onset of GBM, offering potential implications for the early prognostication of bevacizumab therapy outcomes.

Association of maternal angiotensin II type 1 and type 2 receptor combination genotypes with susceptibility to early-onset preeclampsia.

Allelic variations affecting the activity of the maternal renin-angiotensin system may play a role in the development of hypertensive disorders of pregnancy like preeclampsia, its more severe early-onset form, and intrauterine growth restriction. We examined the association of common allelic variants of angiotensin II type 1 and type 2 receptor genes (AT1R and AT2R) sorted in five AT1R/AT2R receptor combination genotype groups with susceptibility to early-onset preeclampsia (EOP). The occurrence of AT1R (A1166C) and A2TR (C3123A) alleles in wild type (AA, CC), heterozygous (A/C, C/A), and homozygous (C/C, A/A) states was recorded in 84 women with a history of EOP and 84 age-matched controls sorted in five AT1R/AT2R receptor combination genotype (wild type: AA/CC, one mutant: AA/CA, AC/CC, two mutant: AC/CA, AA/AA, CC/CC, three mutants: AC/AA, CC/CA and four mutant: CC/AA) groups, by polymerase chain reaction-RFLP analysis. Three mutant receptor combination genotype carriers were more common in women with a history of EOP than in controls (26.18% vs. 4.76%, p = 0.003, OR = 8.25). Receptor combination genotyping may be of clinical value in: (a) maternal prediction of susceptibility to EOP, (b) disease subtyping for directed studies with receptor signaling antagonists, (c) the broader study of hypertension.

Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle.

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus. In Australia, leishmaniasis is an imported disease that is presenting itself at increased rates because of international travel, the influx of immigrants, and deployment of military operations to endemic regions. Although Leishmania species are morphologically indistinguishable, there is a strong correlation between some causative species of leishmaniasis and the subsequent response to the treatments available and patient outcome. Consequently, identification of the infective species is imperative as misidentification can result in the administering of an ineffective drug. The aim of this study was to develop a simple diagnostic tool with high sensitivity and specificity, which is capable of detecting the presence of the parasite and accurately differentiating the causative species in question. Using the advantageous properties of the maxi-circle kinetoplast DNA, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) targeting the ND7 gene was developed for the analysis of imported cases of human leishmaniasis in Australia. Designed as a dual analysis, concurrent PCR of Leishmania maxi-circle DNA and digestion with two separate enzymes (NlaIII and HpyCH4IV), this study provides an appraisal on 24 imported cases of leishmaniasis between 2008 and 2017. Five Leishmania species were reported, with members of the Viannia subgenus being the most common. The implementation of novel diagnostic procedures for leishmaniasis such as the one reported here is needed to establish a gold standard practice for the diagnosis and treatment of leishmaniasis.

Rapid and Reliable One-Step ABO Genotyping Using Direct Real-Time Allele-Specific PCR and Melting Curve Analysis Without DNA Preparation

ABO genotyping is a molecular diagnostic technique important for transfusion and transplantation in medicine, and human identification in forensic science. Because ABO genotyping are labor intensive and time consuming, the genotyping cannot be firstly used to resolve the serological ABO discrepancy in blood bank. For rapid one-step ABO genotyping, we developed direct, real-time, allele-specific polymerase chain reaction (PCR), and melting curve analysis (DRAM assay) without DNA preparation. In DRAM assay, we used a special PCR buffer for direct PCR, a rapid RBC lysis buffer, white blood cells as template without DNA preparation, allele-specific primers for discriminating three ABO alleles (261G/del, 796C/A, and 803G/C), and melting curve analysis as a detection method. There was 100% concordance among the results of ABO genotyping by the DRAM assay, serologic typing, PCR-RFLP and PCR-direct sequencing of 96 venous blood samples. We were able to reduce the number of manual steps to three and the hands-on time to 12min, compared to seven steps and approximately 40min for conventional ABO genotyping using allele-specific PCR with purified DNA and agarose gel electrophoresis. We have established and validated the DRAM assay for rapid and reliable one-step ABO genotyping in a closed system. The DRAM assay with an appropriate number of allele-specific primers could help in resolving ABO discrepancies and should be valuable in clinical laboratory and blood bank.

Confronting two biomolecular techniques to detect NRF2 gene polymorphism biomarkers.

Aim:Gene polymorphism biomarkers identify individual susceptibility to environmental and occupational hazards. The conventional approach considers polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP), a reliable but expensive and time-consuming two-step procedure. Therefore we evaluated the simpler method confronting two-pair primers (CTPP)–PCR for its robustness and applicability to epidemiologic studies.Materials & methods:We compared CTPP–PCR and PCR–RFLP techniques to detect two NRF2 polymorphisms in a set of biological samples.Results:CTPP–PCR produced contradictory results and required the orthogonal technique for confirming the data.Conclusion:In contrast to PCR-RFLP, CTPP–PCR of NRF2 polymorphisms resulted in ambiguous genotyping which strongly jeopardized heterozygosis classification. The necessity of long optimization and control procedures nullified the potential advantages of CTPP–PCR in terms of costs and time.

Morphometric phenotypes and molecular identification of <em>Fasciola</em> species isolated from cattle in Ilorin, North-Central Nigeria

Fascioliasis is a serious infectious parasitic disease of human and domestic ruminants. It is caused by the common liver flukes Fasciola hepatica and Fasciola gigantic and it is of public health concern in some countries of the world. Species-level identification of these species using only morphological methods is unconvincing, unreliable and sometimes difficult. Molecular methods enhance the prospects of species-level identifications using a well established and highly reliable DNA-based approach. We used PCR-RFLP analysis for precise identification of Fasciola species collected from Nigeria. One hundred Fasciola adult worms were collected from selected abattoirs in Ilorin, North-central Nigeria. The parasitic worms were first identified by morphometric analysis using AmScope digital camera fastened to the ocular eye piece of the light microscope. For the molecular identifications, genomic DNA was extracted, Polymerase Chain Reaction (PCR) technique and RFLP were used to characterise the Fasciola adult worms. Morphometric data showed that 70 out of the 100 Fasciola adult worms have characteristic features similar with that of F. gigantica as described from previous studies while the remaining 30 adult worms have characteristics that deviate from that of F. gigantica but did not confirm with characteristic features of F. hepatica. Agarose gel electrophoresis result confirmed successful amplification of two gene regions of Sodium Dehydrogenase I (ND1) from mitochondrial DNA of Fasciola spp. The PCR-RFLP analysis did not show any noticeable genetic variation in the Fasciola adult worms. We suggest more studies to be done using DNA sequence analysis and other molecular markers such as random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) to better understand the genetic variability and population structure within Fasciola spp. in Nigeria.

Vitamin D receptor gene TaqI and Apal polymorphisms and steroid responsiveness in childhood idiopathic nephrotic syndrome.

BackgroundVitamin D activity is controlled by vitamin D receptors (VDRs), which are affected by different genetic polymorphisms, including TaqI and Apal restriction fragment length polymorphisms (RFLPs), which have been reported to be associated with several diseases. The aim of this study was to determine the frequency and the association of VDR gene polymorphisms with idiopathic nephrotic syndrome (INS) and steroid responsiveness in Kuwaiti children.Subjects and methodsGenotypes of the VDR TaqI gene polymorphism and the Apal gene polymorphism were analyzed using polymerase chain reaction-RFLP in 78 INS patients and 56 matched controls.ResultsA total of 78 INS (62 steroid sensitive [SS] and 16 steroid resistant [SR]) patients with a mean age of 6.5±3.1 years were studied. Male:female ratio was 2:1. The TT genotype of VDR–TaqI polymorphism was detected in 41% of the INS patients compared to 42% of the controls (P=0.816). The heterozygous TC genotype was detected in 33% of INS patients compared to 46% of the controls (P=0.462). The CC genotype was detected in 25.6% of INS patients and 21% of the controls (P=0.719). The C-allele frequency, in its homozygous and heterozygous forms, was 71% in INS patients compared to 63% in the controls (P=0.342). Similarly, no significant difference was detected in terms of VDR–Apal polymorphism in INS patients compared to the controls for all the three genotypes (P=0.76, P=0.207, and P=0.364, respectively, for GG, GT, and TT genotypes). The T-allele frequency, in its homozygous and heterozygous forms, was 89% in INS patients compared to 93% in the controls (P=0.076). No significant difference was found in any of the allele frequencies between SS and SR subgroups when compared with each other or when compared to the controls.ConclusionOur data do not support the use of VDR–TaqI or –Apal gene polymorphisms as genetic markers of INS nor do they predict steroid responsiveness in children with the disease.

Survey of chloroquine-resistant mutations in the Plasmodium falciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen

Malaria is still a major public health problem in Yemen. More than 95% of the malaria cases are due to Plasmodium ‎falciparum‎. Recently in Yemen, the antimalarial treatment policy was changed from chloroquine (CQ) to artemisinin combination therapy (ACTs). However, CQ is still available and prescribed in the Yemeni market. The persistence of CQ resistance will be prolonged if the shift to ACT and the simultaneous withdrawal of CQ are not rigorously implemented. The aim of the current survey is to detect chloroquine-resistant mutations in P. falciparum chloroquine-resistance transporter (pfcrt) and P. falciparum multi-drug resistance-1 (pfmdr1) genes. These data will be important for future monitoring and assessment of antimalarial drug policy in Yemen. Blood specimens were collected from 735 individuals from different districts of the Hadhramout province, Yemen by house-to-house visit. Mutation-specific nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR–RFLP) methods were used to investigate the mutations in the pfmdr1(codons 86 and 1246) and pfcrt (codons 76, 271, 326, 356 and 371) genes. The overall prevalence of pfcrt mutations at codons 76, 271, 326 and 371 were 50.4%, 58.7%, 54.3% and 44.9%, respectively. All isolates had wild-type pfcrt 356 allele. The majority of pfmdr1 86 alleles (83.3%) and all pfmdr1 1246 alleles were wild type. There was no association between pfcrt mutations and symptomatology, gender and age groups. In conclusion, point mutations in codons 76, 271, 326 and 371 of pfcrt of P. falciparum are high suggesting a sustained high CQ resistance even after 4 years of shifting to ACTs. These findings warrant complete withdrawal of CQ use from the Yemeni market for P. falciparum and careful usage of CQ for treating Plasmodium vivax.

Vitamin D receptor gene polymorphisms (TaqI and ApaI) in relation to 25-hydroxyvitamin D levels and coronary artery disease incidence

Context/objective: Previous studies have illustrated the association of the ApaI and TaqI polymorphisms of the vitamin D receptor gene, located in non-coding and coding regions, respectively, with diseases such as cancer and cardiovascular disease; however, investigating such association in Egyptian patients with coronary artery disease (CAD) has never been formerly attempted. Materials and methods: Male patients (n = 137), 35–50 years of age, with verified CAD, were recruited alongside age- and sex-matched controls (n = 58). Genotyping and 25-hydroxyvitamin D [25(OH)D] measurement were performed by polymerase chain reaction RFLP and HPLC, respectively. Results: Comparison of the genotypic distribution of both the TaqI and ApaI polymorphisms between patients and controls yielded insignificant results (p = 0.55 and 0.7, respectively). Comparison of the allelic distribution of both polymorphisms also yielded insignificant results. The TaqI polymorphism was not found to predict 25(OH)D levels, whereas the wild-type genotype of the ApaI polymorphism was associated with greater levels of 25(OH)D (p = 0.02), taking all subjects into consideration. Discussion/conclusion: This study presents the ApaI and TaqI polymorphisms as non-influencing players in the pathogenesis of CAD in Egyptian males and the ability of only the ApaI polymorphism to predict 25(OH)D levels, thus warranting further investigations of the triangular relationship between the polymorphisms, 25(OH)D and CAD incidence.

Genetic relationship in mulberry (Morus L.) inferred through PCR–RFLP and trnD-trnT sequence data of chloroplast DNA

Ten universal primer pairs of the plant chloroplast genome were used to amplify the chloroplast DNA (cpDNA) non-coding regions in eight mulberry (Morus spp.) genotypes, including M. mongolica, M. bombycis, M. alba, M. atropurpurea and M. multicaulis. Subsequently, the polymerase chain reaction (PCR) products were digested by seven restriction enzymes and the trnD-trnT fragment for sequence alignment, and the variations were expected to provide the genetic information for system classification. The results from this study showed that: (1) 10 cpDNA primer pairs could be used for successful amplification in the tested materials, with approximately 17.1 kb of the chloroplast genome analysed. The 152 marker loci were detected by 70 primer/restriction endonuclease combinations, among which the trnD-trnT non-coding region digested by AluI, HinfI, MvaI and RsaI was detected by visible fragment length variation in different genotypes of the genus Morus. (2) eight Morus L. genotypes were divided into two groups based on the digesting pattern discrepancy through cpDNA. The M. multicaulis genotypes displayed diversity on an intraspecies level. ‘Nongsang No.12’ was identical with the female parent ‘Beiqu No.1’ (M. atropurpurea) in the surveyed sequence, but different from the male parent ‘Tongxiangqing’ (M. multicaulis), suggesting that the cpDNA was maternal inheritance in Morus L. (3) There were two deletion fragments (451–456 bp; 840–863bp) and six base point mutations in the trnD-trnT region based on homologous sequence alignment. The sequence of trnD-trnT in the cpDNA of mulberry could provide more genetic information for phylogenetic analysis and pedigree identification.

Staphylococcal species associated with bovine mastitis in the North West of Iran: Emerging of coagulase-negative staphylococci

Staphylococcal mastitis is a major and costly problem of dairy cattle all over the world. The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 7 dairy herds located in 4 different areas of East and West Azerbaijan provinces, Iran. Of the 158 mastitic milk samples collected, 113 staphylococcal isolates were identified (71.5%) on the basis of cultural and biochemical features as well as by genus specific PCR. Then, species level identification of staphylococcal isolates was carried out using restriction fragment length polymorphism (RFLP) analysis of the gap gene (933bp). On the basis of polymerase chain reaction-RFLP, 10 different patterns were identified. Of 113 isolates, 5 (4.4%) were Staphylococcus aureus and 108 (95.6%) were coagulase-negative staphylococci (CoNS). Overall, nine different species of CoNS were identified as: 44 Staphylococcus haemolyticus (40.7%), 17 Staphylococcus chromogenes (15.7%), 11 Staphylococcus epidermidis, Staphylococcus warneri and Staphylococcus cohnii each (10.2%), 6 Staphylococcus simulans (5.5%), 4 Staphylococcus hominis (3.7%), 3 Staphylococcus capitis (2.7%) and 1 Staphylococcus xylosus (0.9%). S. haemolyticus, S. chromogenes and S. warneri were the only species identified from clinical mastitis. No significant difference in staphylococcal IMI was found among the studied herds and regions. This study demonstrated that CoNS, especially S. haemolyticus and S. chromogenes, were predominant and thus be considered as emerging pathogens causing mastitis in the North West of Iran. Our results also revealed that the gap PCR-RFLP was useful for identifying staphylococcal isolates derived from bovine mastitis at species level.

Role of genetic variants of deleted in colorectal carcinoma (DCC) polymorphisms and esophageal and gastric cancers risk in Kashmir Valley and meta-analysis

Genetic alterations in the deleted in colorectal carcinoma (DCC) gene have been a priori reported to associate with metastasis in variety of human cancers. We investigated the association between potentially functional SNPs in DCC and susceptibility to esophageal (EC) and gastric (GC) cancers in Kashmir Valley. We genotyped two SNPs DCC rs714 (A>G) and DCC rs2229080 (C>G) of DCC in 135 EC patients, 108 GC patients, and 195 controls matched by age and sex in Kashmir Valley by polymerase chain reaction-RFLP method. Risk for developing EC and GC was estimated by binary logistic regression by using SPSS. We also performed a meta-analysis on DCC rs714 (A>G) and evaluated the association between the DCC rs714 (A>G) polymorphisms and cancer risk. A significant difference in DCC rs714 (A>G) genotype distribution between EC and GC cases and corresponding control groups was observed (odds ratio (OR) = 1.92; P = 0.03; P-trend = 0.04; false discovery rate (FDR) Pcorr = 0.03: OR = 2.15; P = 0.02; P-trend = 0.01; FDR Pcorr = 0.03). But no such association was observed in DCC rs2229080 (C>G). Further, DCC rs714 (A>G) AA genotype showed significantly increased risk for both gastric squamous cell carcinoma (OR = 5.63; P = 0.02; FDR Pcorr = 0.01) and gastric adenocarcinoma (OR = 2.15; P = 0.02; FDR Pcorr = 0.01). Smoking and salted tea are independently associated with both EC and GC, but gene-environment interaction did not further modulate the risk. Meta-analysis also suggested both independent and overall association of DCC rs714 (A>G) polymorphism with cancer (P = 0.000). In conclusion, genetic variations in DCC rs714 (A>G) modulate risk of EC and GC in high-risk Kashmir population.

The effects of polymorphisms in 7 candidate genes on resistance to Salmonella Enteritidis in native chickens

Salmonella enterica serovar Enteritidis infection is a common concern in poultry production for its negative effects on growth as well as food safety for humans. Identification of molecular markers that are linked to resistance to Salmonella Enteritidis may lead to appropriate solutions to control Salmonella infection in chickens. This study investigated the association of candidate genes with resistance to Salmonella Enteritidis in young chickens. Two native breeds of Malaysian chickens, namely, Village Chickens and Red Junglefowl, were evaluated for bacterial colonization after Salmonella Enteritidis inoculation. Seven candidate genes were selected on the basis of their physiological role in immune response, as determined by prior studies in other genetic lines: natural resistance-associated protein 1 (NRAMP1), transforming growth factor β3 (TGFβ3), transforming growth factor β4 (TGFβ4), inhibitor of apoptosis protein 1 (IAP1), caspase 1 (CASP1), lipopolysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), and TNF-related apoptosis-inducing ligand (TRAIL). Polymerase chain reaction-RFLP was used to identify polymorphisms in the candidate genes; all genes exhibited polymorphisms in at least one breed. The NRAMP1-SacI polymorphism correlated with the differences in Salmonella Enteritidis load in the cecum (P = 0.002) and spleen (P = 0.01) of Village Chickens. Polymorphisms in the restriction sites of TGFβ3-BsrI, TGFβ4-MboII, and TRAIL-StyI were associated with Salmonella Enteritidis burden in the cecum, spleen, and liver of Village Chickens and Red Junglefowl (P < 0.05). These results indicate that the NRAMP1, TGFβ3, TGFβ4, and TRAIL genes are potential candidates for use in selection programs for increasing genetic resistance against Salmonella Enteritidis in native Malaysian chickens.

Prevalence ofToxoplasma gondiifrom Free-Range Chickens (Gallus domesticus) from Addis Ababa, Ethiopia

Prevalence of Toxoplasma gondii in free-range chickens (Gallus domesticus) is a good indicator of the environmental contamination with oocysts because chickens become infected mainly by feeding from ground, feed, or soil contaminated with oocysts. The seroprevalence of T. gondii antibodies in 125 free-range chickens from the Addis Ababa, Ethiopia, was determined. Antibodies to T. gondii were assayed by the modified agglutination test; 48 of 125 (38.4%) chickens were seropositive, with titers of 1:5 in 14, 1:10 in 12, 1:20 in 14, 1: 40 in 3, 1: 80 in 1, 1:160 in 1, 1:320 in 1, and ≥1:640 in 2 chickens. The hearts of 115 chickens were bioassayed for T. gondii infection. Hearts of 72 seronegative (modified agglutination test [MAT] < 1:5) chickens were pooled in 4 groups (20 + 18 + 19 + 15) and fed to 4 T. gondii -free cats; none of these 4 cats shed oocysts in their feces examined 3-21 days after feeding chicken tissues. Hearts of 43 seropositive chickens (MAT ≥ 1:5) were bioassayed individually in mice. Toxoplasma gondii was isolated from only 1 chicken, with a MAT titer of 1:80. This isolate was designated TgCKEt1 and was not pathogenic for outbred mice. Restricted fragment length polymorphism (RFLP) genotyping using 10 loci indicated the TgCKEt1 was ToxoDB polymerase chain reaction-RFLP genotype #1 (Type II clonal). Results of this study indicate very low environmental contamination with T. gondii oocysts around Addis Ababa.

A Comparison of Microscopy and PCR Diagnostics for Low Intensity Infections of Haemosporidian Parasites in the Siberian TitPoecile cinctus

This study compares the sensitivity of two methods for diagnosing avian haemosporidian parasite (Plasmodium, Haemoproteus and Leucocytozoon) prevalence in Siberian tits Poecile cinctus breeding in northern Finland: a polymerase chain reaction (PCR)-based method and microscopy examination of blood smears. During molecular analysis, we used PCR screening and RFLP as a post-PCR diagnostic step. PCR screening detected haemosporidian parasites in the blood of 38 out of 40 (95%) breeding Siberian tits. Microscopy examination of blood smears and PCR diagnostics showed the same prevalences for Eeucocytozoon spp. infections. In contrast, prevalences of Haemoproteus spp. and Plasmodium spp. determined by molecular methods were significantly higher than the prevalence determined by microscopy screening. However, habitat and sex effects on the total count of parasites, Plasmodium spp., Haemoproteus spp. and Eeucocytozoon spp. counts appeared to be similar when assessed with either screening method, giving the same in...

Association of angiotensin-converting enzyme (ACE) gene polymorphism with elevated serum ACE activity and major depression in an Iranian population

Genetic factors contribute substantially to the likelihood of developing major depressive disorder (MDD). The importance of renin–angiotensin system (RAS) elements in cognition and behaviour and their involvement in aetiology and treatment of depression imply that RAS gene polymorphism(s) associated with RAS overactivity might also be associated with depression. In the present study, genotype and allele frequencies of six common polymorphisms of genes encoding for RAS components were determined in DNAs extracted from venous blood of 191 depressed and 104 healthy individuals using polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and serum angiotensin-converting enzyme (ACE) activity was assayed using a high-performance liquid chromatography (HPLC) method. The results showed, for the first time, that GG genotype of ACE A2350G was significantly associated with MDD among Iranian participants (P=0.001; odds ratio (OR)=6.2; 95% confidence interval (CI)=2.1–18.3). Significant higher serum ACE activity (P=0.0001) as well as higher diastolic blood pressure (P=0.036) were observed in depressed patients compared to the healthy control group. Depressed patients carrying GG genotype of the A2350G polymorphism had a significantly higher serum ACE activity (P=0.02) than individuals with either AA or AG genotype. In conclusion, this study supports the hypothesis of RAS overactivity in depression in that the genotype associated with higher serum ACE activity in an Iranian population was also associated with MDD.

Influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to organophosphate pesticides

Previous studies have revealed that organophosphate pesticides (OPs) are primarily metabolized by xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticides-exposed workers. Present study was designed to determine the influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to OPs. We examined 268 subjects including 134 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using alkaline comet assay and genotyping was done using individual polymerase chain reaction (PCR) or polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Acetylcholinesterase and paraoxonase activity were found to be significantly lowered in workers as compared to control subjects which were analyzed as biomarkers of toxicity due to OPs exposure ( p < 0.001). Workers showed significantly higher DNA tail moment (TM) compared to control subjects (14.32 ± 2.17 vs. 6.24 ± 1.37 tail % DNA, p < 0.001). GSTM1 null genotype was found to influence DNA TM in workers ( p < 0.05). DNA TM was also found to be increased with concomitant presence of NAT2 slow acetylation and CYP2C9*3/ *3 or GSTM1 null genotypes ( p < 0.05). DNA TM was found increased in NAT2 slow acetylators with mild and heavy smoking habits in control subjects and workers, respectively ( p < 0.05). The results of this study suggest that GSTM1 null genotypes, and an association of NAT2 slow acetylation genotypes with CYP2C9*3/ *3 or GSTM1 null genotypes may modulate DNA damage in workers occupationally exposed to OPs.

Cholesteryl Ester Transfer Protein Taq1B (g.5454G>A) Gene Polymorphism in Primary Combined Hyperlipidemia in the Egyptian Population

Objective: To study the relationship between Taq1B polymorphism of the cholesteryl ester transfer protein (CETP) gene and lipid profile in the Egyptian population. A base substitution from G (B1) to A (B2) in intron 1 of the gene leads to 1 of 3 variants, B1B1, B1B2, or B2B2, at the Taq1B site (5454G>A) (Mutalyzer 2.0 B-6). Materials and Methods: Lipid profile parameters and CETP Taq1B polymorphisms were studied in 30 patients with primary combined hyperlipidemia and in 28 control subjects. The lipid profile was measured. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to study Taq1B polymorphisms. Results: The B2B2 genotype was significantly lower in the patient group (13.3%) than it was in the control group (35.7%). No significant difference was found in allelic distribution between groups. In the patient group, the B2B2 genotype showed a higher serum high-density lipoprotein cholesterol (HDL-C) and lower total cholesterol (TC)/HDL ratio than B1B1 and B1B2. In the control group, triglycerides (TGs), TC/HDL ratio, and low-density lipoprotein (LDL) levels were lower in the B2B2 genotype than other genotypes. Conclusion: There was a suggested association between Taq1B polymorphism of the CETP gene and lipid profile changes, with the B2B2 genotype being protective against hyperlipidemia and atherosclerosis. * CETP : cholesteryl ester transfer protein PCR : polymerase chain reaction RFLP : restriction fragment length polymorphism HDL-C : high-density lipoprotein cholesterol TC : total cholesterol LDL : low-density lipoprotein CHD : coronary heart disease VLDL : very-low-density lipoprotein AST : aspartate transaminase ALT : alanine transaminase TSH : thyroid-stimulating hormone TG : triglyceride dATP : deoxyadenosine triphosphate cDTP : deoxycytidine triphosphate dGTP : deoxyguanosine triphosphate dTTP : deoxythymidine triphosphate ANOVA : analysis of variance CVD : cardiovascular disease

Frequency of Uridine Monophosphate Synthase Gly213Ala Polymorphism in Caucasian Gastrointestinal Cancer Patients and Healthy Subjects, Investigated by Means of New, Rapid Genotyping Assays

Uridine monophosphate synthase (UMPS) is a fundamental enzyme in pyrimidine synthesis. A single-nucleotide polymorphism, a G-C transversion at the 638th nucleotide, was demonstrated to increase UMPS activity and suggested to have clinical effects. The aims of this study were to set up simple genotyping methods and investigate the UMPS 638G>C polymorphism in the Caucasian population. Two hundred forty-one patients with gastrointestinal cancers and 189 healthy subjects were enrolled. Genomic DNA was extracted from peripheral blood. A polymerase chain reaction-restriction fragment length polymorphism (RFLP) method was implemented using a forward primer incorporating a mismatched base to produce an artificial restriction site and BsrI restriction enzyme digestion; a denaturing high performance liquid chromatography (DHPLC) method was developed to further speed up UMPS genotyping. A 153 bp UMPS gene fragment was successfully amplified and analyzed in all samples. RFLP and DHPLC results showed a 100% match and where confirmed by direct sequencing. UMPS genotype distribution was similar in patients with cancer and control subjects. Although no association was detected between UMPS variants and gastrointestinal cancer risk in Caucasians, polymerase chain reaction-RFLP with BsrI digestion and DHPLC set up at 59°C are reliable and cost-effective methods to genotype UMPS.