Polymerase Chain Reaction With Restriction Fragment Length Polymorphism Research Articles

Analysis of MMP-2-735C/T (rs2285053) and MMP-9-1562C/T (rs3918242) Polymorphisms in the Risk Assessment of Developing Lung Cancer

Matrix metalloproteinase (MMP)-2 and -9 are gelatinases which are capable of degrading type IV collagen and have been linked to cancer invasion and metastatic development. MMP-2 and MMP-9 gene polymorphisms may affect their biological function, and thus their role in cancer development and progression. We analyzed the association of the polymorphism frequencies of MMP-2-735C/T and MMP-9-1562C/T with MMP-2 and MMP-9 serum concentrations, as well as their potential effects in lung cancer patients. We conducted a retrospective, case-control study consisting of 112 lung cancer patients and 100 healthy individuals from a Caucasian population in Poland. Polymerase chain reaction with restriction fragment length polymorphism (PCR/RFLP) and electrophoresis was used to genotype genomic DNA from whole blood samples. MMP-2 and MMP-9 serum concentrations were then determined using ELISA. For statistical analysis, Statistica version 13 from TIBCO Software Inc. was utilized with a significance level <0.05. Logistic regression analysis revealed that MMP-2-735CC (OR = 5.39; 95% CI = 0.62–47.17; p = 0.238504) and -735CT genotype (OR = 7.22; 95% CI = 0.78–67.14; p = 0.072836), as well as MMP-9-1562CC (OR = 1.45; 95% CI = 0.31–6.70; p = 0.757914) and -1562CT genotype (OR = 1.60; 95% CI = 0.33–7.83; p = 0.548801) were associated with a higher risk of lung cancer. There were statistically significant differences observed in the MMP-2 concentration between individuals with the -735CC genotype and the -735CT genotype (non-smoking control: 204.04 ng/mL vs. 237.00 ng/mL, respectively, p = 0.041479; adenocarcinoma patients: 157.69 ng/mL vs. 126.37 ng/mL, respectively, p = 0.013222), as well as differences in the MMP-9 concentration between individuals with the -1562CC genotype and the -1562CT genotype (smoking control: 385.67 ng/mL vs. 562.80 ng/mL, respectively, p = 0.000936; patients with other lung neoplasms: 821.64 ng/mL vs. 928.88 ng/mL, respectively p = 0.023315). The role of MMP-2-735C/T and MMP-9 -1562C/T polymorphisms in an increased risk of lung cancer cannot be dismissed. Specific genotypes affect MMP-2 and MMP-9 concentrations in both lung cancer patients and healthy controls, which may thereby increase lung cancer risk, disease aggressiveness, and patient survival outcomes.

Vascular endothelial growth factor (VEGF-2578 C > A) gene polymorphism as a genetic biomarker for breast cancer: A case control study

Breast cancer (BC) is one of the most frequently diagnosed cancers among Saudi women. Vascular endothelial growth factor (VEGF) is a key mediator of breast cancer angiogenesis. Hence, the present study was aimed to investigate the genetic association of VEGF (-2578 C > A, rs699947) polymorphism with BC among Saudi women. The present study includes 104 BC cases and 100 controls. Polymorphic analysis was carried out adopting polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) method. The homozygous variant (AA) (RR = 3.96, EF = 0.583, p = 0.005) and allele A (RR = 1.56, EF = 0.206, p = 0.03) of VEGF (-2578C > A) gene were significantly (p < 0.05) higher in the BC cases when compared to the controls. The recessive genotypes' combination (CC + CA) has shown a strong association (p = 0.003) with BC. Further, AA genotype has shown a strong association (p = 0.009) with BC in patients >40 years of age. AA genotype was significantly (p = 0.023) associated with advanced (T4) stage and high histologic tumor grades II (p = 0.015) and III (p = 0.006) of BC patients as well. Therefore, the homozygous variant and allele A of VEGF (−2578 C > A) gene polymorphism could be an associative genetic risk factor for BC in Saudi population. Regulatory region polymorphism in VEGF may help better to predict the risk of the BC at an early stage and will be assistive in the development of new diagnostic markers and treatment strategies.

Genetic Polymorphisms in Transforming Growth Factor-β, Interferon-γ and Interleukin-6 Genes and Susceptibility to Behcet's Disease in Saudi Population.

BackgroundBehcet’s disease (BD) is a complex, inflammatory, immune-mediated multi-systemic disease of unknown etiology. Cytokines play major roles in the pathophysiology of BD, and its production may be affected by polymorphism in cytokine genes. Hence, the present study was planned to investigate any possible association between the polymorphism in TGF-β, IFN-γ, and IL-6 genes and BD in the Saudi population.Materials and MethodsThe present study includes 79 BD cases and 117 age-matched controls. Polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation systems (ARMS) PCR methods were used for polymorphic analysis.ResultsThe heterozygous (CT) and (CT+TT) combined genotypes of TGF-β (509-C/T), heterozygous (AT), variant (TT), and (AT+TT) combined genotypes of IFN-γ (874-A/T) were significantly (P<0.05) associated with BD in the Saudi population. No significant differences were observed for IL-6 (174-G/C) genotypes and alleles between BD cases and controls. Gender does not reflect any significant genotypic and allelic association with males and females.ConclusionCT genotype of TGF-β, and AT and TT genotypes of IFN-γ could be associative genetic risk factors for BD in Saudis. Regulatory region polymorphism in cytokines gene can increase inflammation and deregulated immune response, which could be risk factor for BD.

Effects of probiotics or broccoli supplementation on Helicobacter pylori eradication with standard clarithromycin-based triple therapy.

Background/AimsThe eradication failure rate of standard triple therapy (proton pump inhibitor, clarithromycin, and amoxicillin) for Helicobacter pylori infection has increased owing to antibiotic resistance in Korea. We assessed whether Saccharomyces boulardii probiotic or broccoli sprout extract sulforaphane supplementation could increase the H. pylori eradication rate and/or reduce antibiotic-associated adverse events.MethodsA total of 217 patients with H. pylori-positive chronic gastritis or peptic ulcer disease were recruited. Clarithromycin resistance was assessed in all patients by testing for A2142G and A2143G point mutations in H. pylori 23S rRNA using a dual-priming polymerase chain reaction (PCR) oligonucleotide. Thirty-four patients (17.3%) were clarithromycin-resistant and were excluded from the study. Finally, 183 patients with infections not resistant to clarithromycin were randomly assigned to triple therapy only (group A, n = 61), triple therapy plus probiotics (group B, n = 61), or triple therapy plus sulforaphane (group C, n = 61) groups. CYP2C19 polymorphisms were examined at position G681A of exon 5 and G636A of exon 4 by PCR with restriction fragment length polymorphism (PCR-RFLP) analysis. H. pylori eradication was assessed by 13C-urea breath test 4 weeks after treatment completion.ResultsThe eradication rates were similar among the groups both in the intention-to-treat (A = 85.2%, B = 89.6%, and C = 81.6%) and per-protocol (A = 89.2%, B = 86.8%, and C = 96.3%) analyses. The frequencies of overall adverse events in the groups also did not differ (A vs. B: p = 0.574; A vs. C: p = 1.000).ConclusionsProbiotic or sulforaphane with triple therapy for H. pylori infection neither increased the eradication rate nor reduced the occurrence of adverse events.

Carrier frequency of autosomal recessive disorders (BC, BLAD, FXID and CVM) in Holstein cows in Jalisco, Mexico

ABSTRACT: The hereditary autosomal recessive disorders bovine citrullinemia (BC), bovine leukocyte adhesion deficiency (BLAD), factor XI deficiency (FXID), and complex vertebral malformation (CVM) have affected dairy cattle breeding significantly around the world. This study examined the carrier frequency of BC, BLAD, FXID, and CVM autosomal recessive disorders in Bos taurus Holstein cows bred in the Altos Norte region of the state of Jalisco, Mexico. We extracted DNA from 408 random samples of peripheral blood, and then used polymerase chain reaction (PCR) to identify insertion mutations for FXID, and PCR with restriction fragment length polymorphism (PCR-RFLP) for CVM, BC and BLAD. We visualized the PCR products using agarose gel electrophoresis stained with GelRed®. We found that 100% of wild-type (N/N) allele homozygous animals for genes CD18, ASS, and FXI were free of the mutations for BLAD, BC and FXID respectively. For gene SLC35A3 we estimated total carrier frequency of 10.3% and allele frequency of 5%.

Association of P2X7 gene common polymorphisms with pulmonary tuberculosis in Lur population of Iran

BackgroundDifferent genetic and environmental factors are associated with susceptibility to pulmonary tuberculosis (TB) in different individuals of different populations. Based on previous studies role of P2X7 gene common polymorphisms in susceptibility to pulmonary TB was associated with ethnicities. AimWe intend to perform this study on genetic reservoir (gene pool) of Lur population of western Iran. MethodsFor the present case-control study, 100 unrelated pulmonary TB patients and 100 unrelated controls were enrolled through convenient sampling. TB confirmation was through smear and culture of sputum. Polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) was used for molecular assay. This study has been approved in the ethic committee of Lorestan University of Medical Sciences with registration number LUMS.REC.1396.253. ResultsAmong the genotypes of polymorphism 1513A/C, AA genotype was associated with susceptibility to pulmonary TB (P = .0001; OR = 4.750) whereas AC genotype was a protecting factor (P = .0001; OR = 0.192). Higher genetic reservoir of A allele was associated with more susceptibility to pulmonary TB (P = .0001; OR = 2.879) whereas C allele was a protecting factor (P = .0001; OR = 0.347). No significant result was found for −762T/C polymorphism. ConclusionIn Lur population of Iran, 1513A/C polymorphism of P2X7 is associated with susceptibility to pulmonary TB. It is suggested that bio-information banks should be established and developed in countries.

Predominance and association risk of Blastocystis hominis subtype I in colorectal cancer: a case control study

BackgroundBlastocystis, a genetically diverse intestinal parasite with controversial pathogenic potential, has increasingly been incriminated for diarrheal illness in immunocompromised individuals including colorectal cancer (CRC) patients. The aim of the current study was to assess the possible association between Blastocystis infection and CRC condition in Makkah, Saudi Arabia (KSA).MethodsStool samples were collected from 80 non-cancer (NC) and 138 cancer subjects including 74 CRC patients and 64 patients with other cancers outside gastrointestinal tract (COGT). Molecularly confirmed Blastocystis isolates were genetically grouped and subtyped using multiplex polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and sequence-tagged site primers-based PCR (PCR-STS), respectively.ResultsBlastocystis hominis were confirmed in 29.7, 25 and 15% among CRC, COGT and NC patients, respectively. Obtained Blastocystis isolates were initially categorized into 2 groups (A and C), which were subsequently subtyped into 3 different subtypes; subtype-I (38%), subtype-II (44%) and subtype-V (22%). Interestingly, subtype-I was the most predominantly detected subtype (54.5%) among CRC patients with a significant association risk (COR 7.548; 95% CI: 1.629–34.987; P = 0.004).ConclusionTo the best of our knowledge, the current study is the first to provide genetic insights on the prevalence of Blastocystis hominis among CRC patients in Makkah, KSA. Moreover, the study suggests for a possible association between subtype-I of Blastocystis hominis and CRC, which could indicate a potential influence of Blastocystis on CRC condition. Further studies are required to confirm this association risk and to investigate the possible underlying mechanism of postulated carcinogenic influence of Blastocystis hominis subtype-I.

Characterization of Toxoplasma gondii isolates from herds of sheep in southern Brazil reveals the archetypal type II genotype and new non-archetypal genotypes

Recent studies have demonstrated that, in Brazil and South America, strains of Toxoplasma gondii are often genotypically and biologically different from those found in countries on other continents. The objective of this study was to genotypically characterize T. gondii isolates from naturally infected sheep in herds in the southern region of the state of Rio Grande do Sul, Brazil, by means of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP). Five T. gondii isolates obtained from sheep in five municipalities in the state of Rio Grande do Sul were used. Application of multilocus PCR-RFLP multilocus using 12 genetic markers (SAG1, 5′3′ SAG2, alt. SAG2, SAG3, BTUB, c22-8, c29-2, GRA6, L358, PK1, APICO and CS3) revealed four different genotypes in the five isolates studied: clonal type II (TgOvBrRS4), type BrIV (TgOvBrRS2 and TgOvBrRS3) and two new non-archetypal genotypes, ToxoDB-RFLP#270 and #271 (TgOvBrRS1 and TgOvBrRS5, respectively). The genotype structure found in the T. gondii isolates from naturally infected sheep in the southern region of Brazil was revealed to have high diversity. This study confirms the presence of rare circulation of the clonal type II genotype in Brazil.

Effects of ApoE gene polymorphism on the efficacy of atorvastatin in the treatment of hyperlipidemia

Objective: To study the role of ApoE gene polymorphism on efficacy of atorvastatin in lowering the lipid and its clinical significance. Methods: A total of 962 patients with hypercholesterolemia were selected between January 1 st and December 31 st 2014. The ApoE genepolymorphism in patients with hyperlipidemia was performed by using polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) method in translational medicine center of Huaihe Hospital. Patients with ApoE genotype E3/3 and E3/4 were selected and treated with atorvastatin 10 mg/d for 4 weeks. Before and after treatment, triglycerides (TG) and total cholesterol (TC) was detected by enzyme colorimetry method. High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were performed by Clearance method. Lipoprotein(a) (Lp(a)) was performed by turbidimetric inhibition immunoassay. ApoE gene expression was performed by real-time PCR. Results: In the 6 gene types, the frequencies of E3/4 and E3/3 were 30.6% (294 cases) and 59.1% (569 cases) respectively. After treatment with atorvastatin, the change percent of TC, LDL-C, HDL-C, TG, Lp(a) in E3/4 and E3/3 group were -(23.0±4.7)% vs -(12.0±3.1)% (P<0.001), -(33.0±4.8)% vs -(20.0±3.9)% (P<0.001), (18.0±3.8)% vs (6.0±2.6)% (P<0.001), -(23.0±3.9)% vs -(13.0±2.7)% (P<0.001), -(21.5±4.5)% vs -(20.9±4.0)% (P=0.054), respectively. ApoE gene expression in E3/3 and E3/4 groups were down-regulated in both groups, and the change in E3/3 group was obvious than that of E3/4 group. Conclusion: After treatment with atorvastatin, levels of lipids and ApoE gene expression in ApoE genotype E3/3 patients decreased, which were more evident than E3/4 patients.

INFLUENCE OF APOE GENOTYPES ON BIOCHEMICAL INDICES OF IRON AND LIPID METABOLISM IN PATIENTS WITH THE ENDOMETRIUM NEOPLASTIC DISEASES AND HEAL THY WOMEN

Objective. APOE genotype association with the level of iron in plasma and the risk of endometrial neoplastic diseases has not been studied. The aim of the work was to analyze the genotypic associations of the apolipoprotein E (APOE) with iron and lipid levels in endometrial hyperplasia (EH)/endometrial cancer (EC) patients and healthy women. Materials and methods. The biochemical blood levels of iron and cholesterol metabolism detected in women d with diagnosed endometrial hyperplasia (53), endometrial cancer (87) and healthy women (70) are determined on an automated analyzer «OLYMPUS AU – 400”. The genotyping was performed with a PCR-RFLP (polymerase chain reaction with restriction fragment length polymorphism) technique. Results. A statistically significant decrease in the levels of serum iron, transferrin saturation, high-density lipoprotein on the one hand, and a statistically significant increase in body mass index, triglycerides, very low density lipoproteins, atherogenic index in patients with endometrial adenocarcinoma was observed. Statistically significant differences in the distribution of E3, E4, E2 allele frequencies between patients and healthy women (P = 0.048) was revealed. E3 allele frequency was reduced, and the E2 allele was increased in patients with EC. It was shown that the serum iron level was significantly increased in EC patients and healthy carriers of the genotype E3/E2 (P= 0,0014 and 0,0363 respectively). There was significantly elevated triglyceride level in carriers of the minor genotypes E2/E4, E4/E4 among patients with EH (0,0389). Conclusions. Genotype E3/E2 compared with other APOE genotypes under study is associated with elevated levels of iron and the risk of endometrial cancer. The association APOE 2/2, 2/4, 4/4 as well as 3/2 with endometrial neoplastic diseases requires further investigation. The data obtained can be the basis for the creation of a marker system for determining a high level susceptibility to malignant disease of the female reproductive system.

The +190 G/A (rs1799864) polymorphism in the C–C chemokine receptor 2 (CCR2) gene is associated with susceptibility to multiple sclerosis in HLA-DRB1*15:01-negative individuals

C–C chemokine receptor 2 (CCR2) is one of the key players involved in the transmigration of mononuclear cells into the central nervous system (CNS) and subsequent development of multiple sclerosis (MS). The aim of the current study was to analyse the association of CCR2 +190 G/A (rs1799864) polymorphism with susceptibility to MS and its influence on the age at onset, severity and neurological disability in MS. CCR2 genotyping was carried out by a polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) in 301 MS patients and 342 healthy controls. Logistic regression analysis suggested a marginally significant association between MS and rs1799864 A allele (AA+GA vs. GG, P=0.047, OR=1.50, 95% CI=1.00–2.25), however, after stratification of study groups for the presence of HLA-DRB1*15:01 risk allele, this association could be found in HLA-DRB1*15:01-negative individuals only (AA+GA vs. GG, P=0.014, OR=1.84, 95% CI=1.13–2.98). Furthermore, there was no association between CCR2 polymorphism and clinical features of MS. In conclusion, our results suggest that CCR2 +190 G/A polymorphism may increase the susceptibility to MS, but its action seems to be restricted to individuals who do not possess the major risk allele HLA-DRB1*15:01.

Tumor necrosis factor alpha promoter polymorphism studies in pregnant women

AimsThe aim of this study was to explore the possible association between the −850C/T polymorphism in the tumor necrosis factor alpha (TNF-α) gene promoter, and pregnancy-associated diseases such as gestational diabetes mellitus (GDM) and preeclampsia (PE), in south Indian women. GDM and PE are common complications that occur during pregnancy and are the leading causes of perinatal mortality. To date, the mechanisms that initiate GDM and PE in humans have remained elusive. MethodsThis prospective case-control study was carried out with 505 pregnant women: 140 women had GDM, and 105 with PE. Remaining 260 women were age- and frequency-matched controls. TNF-α (–C850T) genotyping was determined by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) analysis. ResultWe found no statistically significant difference in the genotypic and allelic distribution between GDM women and controls (for CT + TT vs. CC, χ2 = 0.3919; p = 0.61; Odds Ratio (OR) = 0.76 (95% CI: 0.203–1.876)). No significant differences was observed in the allele and genotype frequency between PE women and controls (for CT + TT vs. CC, p = 0.31; OR = 0.55 (95% CI: 0.171–1.784); T vs. C, p = 0.71; OR = 0.94 (95% CI: 0.680–1.3)). ConclusionFrom our results, we conclude that the (–C850T) promoter polymorphism has no role in the propensity of pregnant women from south Indian populations to develop GDM or PE.

Effects Ala54Thr polymorphism of FABP2 on obesity index and biochemical variable in response to a aerobic exercise training

The purpose of the current study was to investigate whether or not the FABP2 gene polymorphism modulated obesity indices, hemodynamic factor, blood lipid factor, and insulin resistance markers through 12-week aerobic exercise training in abdominal obesity group of Korean mid-life women. A total of 243 abdominally obese subjects of Korean mid-life women voluntarily participated in aerobic exercise training program for 12 weeks. Polymerase Chain Reaction with Restriction Fragment Length Polymorphism (PCR-RFLP) assay was used to assess the FABP2 genotype of the participants (117 of AA homozygotes, 100 of AT heterozygotes, 26 of TT homozygotes). Prior to the participation of the exercise training program, baseline obesity indices, hemodynamic factor, blood lipid factor, and insulin resistance markers were measured. All the measurements were replicated following the 12-week aerobic exercise training program, and then the following results were found. After 12-week aerobic exercise training program, wild type (Ala54Ala) and mutant type (Ala54Thr+Thr54Thr) significantly decreased weight (P > .001), BMI (P > .001), %bf (P > .001), waist circumference (P > .001), WHR (P > .001), muscle mass (wild type p < .022; mutant type P > .001), RHR (P > .001), viseceral adipose area (wild type p < .005; mutant type P > .001), subcutaneous area (P > .001), insulin (wild type p < .005; mutant type P > .001) and significantly increased VO2max (P > .001). And wild type significantly decresed NEFA (P > .05), glucose (P > .05), OGTT 120min glucose (P > .05) and significantly increased HDLC (p > .005). Mutant type significantly decreased SBP (P > .001), DBP (P > .01), TC (P > .01), LPL (P > .05), LDL (P > .001), HOMA index (P > .01). The result of the present study represents that regular aerobic exercise training may beneficially prevent obesity index, blood pressure, blood lipids and insulin resistance markers independent of FABP Ala54Thr wild type and mutant type.

Risk Factors for Rectal Cancer and Methylenetetrahydrofolate Reductase Polymorphisms in a Population in Northeast Thailand

Polymorphisms in methylenetetrahydrofolate reductase (MTHFR) are known to be associated with predisposition for certain cancers. This study aimed to evaluate the effects of lifestyle factors, family history and genetic polymorphisms in MTHFR C677T and A1298C on rectal cancer risk and possible interactions with lifestyle factors in Northeast Thailand. A hospital-based case-control study was conducted during 2002-2006 with recruitment of 112 rectal cancer cases and 242 non-rectal cancer patient controls. Information was collected using a structured-questionnaire. Blood samples were obtained for assay of MTHFR C677T and A1298C genotypes by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) techniques. Associations between lifestyle factors, family history and genetic polymorphisms v.s. rectal cancer risk were assessed using logistic regression analysis. Subjects with frequent and occasional constipation had a higher risk (OR adj.=14.64; 95%CI=4.28-50.04 and OR adj.=2.15; 95%CI=1.14-4.06), along with those who reported ever having hemorrhoids (OR adj.=2.82; 95%CI=1.36-5.84) or a family history of cancer (OR adj.=1.90; 95%CI=1.06-3.39). Consumption of a high level of pork was also associated with risk (OR adj.=1.82; 95%CI=1.05-3.15). Interactions were not observed between MTHFR and other risk factors. This study suggested that the risk factors for rectal cancer in the Thai population are bowel habits, having had hemorrhoids, a family history of cancer and pork consumption.

Polymorphisms of DNA repair genes and risk of squamous cell carcinoma of the head and neck in young adults

Squamous cell carcinoma of the head and neck (HNSCC) most frequently arise in the epithelial tissues of the upper aerodigestive tract. Patients with HNSCC, aged <45 years are categorized as young adults (YA). They are characterized by more severe form of this disease and often lack of classical, causative risk factors (tobacco smoking, alcohol abusing) in comparison to older (typical) patients (OP). The study purpose was to establish an anticipated protective role of DNA repair genes polymorphisms against cancer-causing agents. It was assumed that the polymorphisms in these genes may have a significant role in the etiology of HNSCC in YA. Studies were carried out on three groups: YA group with HNSCC (n = 90), young healthy group without cancer (YH, n = 160) and OP with HNSCC (n = 205). Three polymorphisms in DNA repair genes were analyzed: XPD ex23: A35931C, XRCC1 ex10: G28152A, and XRCC3 ex7: C18067T. The choice of these genes was connected with their involvement in three different DNA repair pathways. Genotyping was carried out by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) technique. Statistical analysis included: calculation of odds ratio (ORs), 95 % confidence intervals (CIs) and p value. There was no significant difference in the distribution of XPD genotypes in YA compared to OP or YH. The XRCC1 AA genotype variant was observed less frequently in HNSCC YA (4.7 %) than in YH and in OP group (17.1 and 10.8 %, respectively). XRCC3 CT genotype variant was observed more frequently in HNSCC YA (61.8 %) than in YH (36.3 %) and this result is statistically significant. This variant was associated with the borderline increased risk of HNSCC development in an early age, however, a similar tendency was not observed in case of double mutated TT variant. The established differences of genotypes distribution do not seem to differentiate substantially YA and OP in head and neck cancer risk.

Correlation between polymorphism in the promoter of DNA methyltransferase-3B and the risk of colorectal cancer

To explore the correlation between the polymorphism in the DNA methyltransferase-3B (DNMT3B) gene promoter single nucleotide polymorphism (SNP)-149C→T (rs2424913) and-579G→T(rs1569686) and the genetic susceptibility to colorectal cancer in Jiangsu population. Genomic DNA was extracted from the leukocyte cell of blood samples collected from 544 colorectal cancer (CRC) patients (including 280 cases of colon cancer and 264 cases of rectal cancer) since January 2009 and July 2010, in a hospital, Jiangsu Province. The same samples were collected from the other 533 control subjects. Polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing analysis were employed to assess the polymorphism of DNMT3B gene promoter-149C→T and-579G→T. For DNMT3B-149C→T, no significant deviation was observed in the genotype distributions of polymorphisms between CRC cases (TT: 98.90% (538/544); CT: 1.10% (6/544)) and controls (TT: 97.75% (521/533); CT: 2.25% (12/533)) (χ(2) = 2.07, P = 0.15). The CC genotype was not detected in either patients or control subjects. The DNMT3B-149CT genotype was not associated with the risk of CRC (adjusted OR = 0.48, 95%CI: 0.18 - 1.30). For DNMT3B-579G→T, the genotype distributions of polymorphisms in CRC patients (TT: 90.07% (490/544); GT: 9.19% (50/544); GG: 0.74% (4/544)) were significantly different from those in control group (TT: 81.80% (436/533); GT: 17.82% (95/533); GG: 0.38% (2/533)) (χ(2) = 15.49, P < 0.05). The results showed that the-579 G allele could significantly decrease the risk of CRC (adjusted OR = 0.50, 95%CI: 0.35 - 0.72) in comparison with the -579 TT genotype. In addition, stratification analysis showed that for DNMT3B-579G→T, the genotype distributions of polymorphisms in colon cancer (TT: 92.50% (259/280); GT: 7.50% (21/280)) were significantly different from those in the controls (TT: 81.80% (436/533); GT: 17.82% (95/533); GG: 0.38% (2/533)) (χ(2) = 13.53, P < 0.05); and similar result was found in rectal cancer (TT: 87.50% (231/264); GT: 10.98% (29/264); GG: 1.52% (4/264)) and controls (TT: 81.80% (436/533); GT: 17.82% (95/533); GG: 0.38% (2/533)) (χ(2) = 5.64, P = 0.018). G allele carriers could decrease the risk of colon cancer (adjusted OR = 0.38, 95%CI: 0.23 - 0.63), and the risk of rectal cancer (adjusted OR = 0.65, 95%CI: 0.42 - 0.99). However, for DNMT3B-149C→T , there were no significant deviation in the genotype distributions of polymorphisms between colon cancer (TT: 98.57% (276/280); CT: 1.43% (4/280)) and controls (TT: 97.75% (521/533); CT: 2.25% (12/533)) (χ(2) = 0.82, P = 0.366); and there was no significant deviation between rectal cancer (TT: 99.24% (262/264); CT: 0.76% (2/264)) and controls (TT: 97.75% (521/533); CT: 2.25% (12/533)) either (χ(2) = 1.89, P = 0.169). Our research demonstrates that the-579 G allele is a potential protective factor for the occurrence of CRC, however, the polymorphism of DNMT3B-149 gene shows no close correlation with the occurrence and development of CRC among Chinese population.

Use of molecular tools in identification of philometrid larvae in fishes: technical limitations parallel our poor assessment of their biodiversity

The usefulness of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and partial sequencing of the cytochrome oxidase I (COI) gene was tested regarding the utility of these techniques in unraveling philometrid life cycles and, in particular, to determine putative paratenic host species. Our focus was to study three species of philometrids commonly found in the estuaries of South Carolina: Philometroides paralichthydis and Philometra overstreeti from the southern flounder, Paralichthys lethostigma and Philometra carolinensis from the spotted seatrout, Cynoscion nebulosus. A total of 473 fish in 19 species known to be potential prey of the spotted seatrout and the southern flounder were dissected. Of all nematode larvae found in the mesenteries of 53 fish of 10 species, 21 specimens were determined to be philometrids using PCR. The use of PCR-RFLP allowed the identification of larvae of P. carolinensis and P. overstreeti in the freshwater goby, Ctenogobius shufeldti, and P. overstreeti in mummichog, Fundulus heteroclitus. However, 12 RFLP profiles could not be matched to control species, thus demonstrating the limitation of this technique in areas where diversity of philometrids is not well known and higher than anticipated. Similarly, COI procedures provided unknown sequences that did not match those of nine philometrid species used as controls. We concluded that although both techniques showed some usefulness and promise, at this point, however, they demonstrate the need of increasing our knowledge of marine and estuarine philometrid biodiversity.

Characterization of glucokinase polymorphisms associated with Maturity-Onset Diabetes of the Young (MODY2) in Jordanian population

Maturity-Onset Diabetes of the Young(MODY) is a monogenic form of Diabetes Mellitus (DM) characterized by an autosomal dominant inheritance, onset usually before 25 years of age and a primary defect in glucose-stimulated insulin secretion. Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. The mutation in the gene encoding GCK results in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. In this study 100 diabetic Jordanian patients with MODY2 phenotype and 150 Normal control subjects were screened for the presence of GCK gene mutations including the missense mutations at position Thr228Ala in exon 7, Gly299Arg in exon 8 and nonsense mutation Ser383Ter in exon 9, utilizing polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) analysis. The results shows no Thr228Ala, Gly299Arg and Ser383Ter mutations were detected in both groups, which was differ from the results obtained for Italian and Caucasian from the Oxford region in UK MODY2 patients. Our data indicated that the previously studied mutations in Italian and Caucasian patients in the GCK gene are not common in MODY Jordanian population, suggesting a racial difference can be found in the frequency of the GCK polymorphism.

A80G polymorphism of reduced folate carrier 1 (RFC1) and C776G polymorphism of transcobalamin 2 (TC2) genes in Down's syndrome etiology

There is evidence that polymorphisms of genes involved in folate metabolism may be associated with higher risk that mothers may bear a Down's syndrome (DS) child. This study therefore had the objective of investigating the A80G polymorphism of the reduced folate carrier 1 (RFC1) gene and the C776G polymorphism of the transcobalamin 2 (TC2) gene as maternal risk factors for DS among Brazilian women. Analytical cross-sectional study with control group, at Faculdade de Medicina de São José do Rio Preto (Famerp). Sixty-seven mothers of DS individuals with free trisomy 21, and 113 control mothers, were studied. Molecular analysis of the polymorphisms was performed by means of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP), followed by electrophoresis on 2% agarose gel. The frequencies of the polymorphic alleles were 0.51 and 0.52 for RFC1 80G, and 0.34 and 0.34 for TC2 776G, in the case and control groups, respectively. Thus, there were no differences between the groups in relation to either the allele or the genotype frequency, for both polymorphisms (P = 0.696 for RFC1 A80G; P = 0.166 for TC2 C776G; P = 0.268 for combined genotypes). There was no evidence of any association between the RFC1 A80G and TC2 C776G polymorphisms and the maternal risk of DS in the sample evaluated.

Hyperhomocysteinemia, Enzyme Polymorphism and Thiobarbituric Acid Reactive System in Children with High Coronary Risk Family History

Objectives: To investigate both the frequency and the genetic background of hyperhomocysteinemia and the frequency of increased plasma thiobarbituric acid reactive system (TBARS) levels in children and adolescents whose parents had premature coronary heart disease (CHD).Methods: The study was performed on children and adolescents aged 4–18 years (105 offspring of parents with CHD before age 45 and 74 referents from families without any evidence of premature atherosclerosis). Fasting serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and total triglyceride (TG) levels were measured by enzymatic methods. Low density lipoprotein cholesterol (LDL-C) level was calculated by the Friedewald formula. Plasma total homocysteine (THCy) level was measured by fluorescence polarisation immunoassay. Plasma TBARS level was determined by fluorimetric method. 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme polymorphism was analyzed by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP).Results: Hyperhomocysteinemia was found in 32 cases and in 4 controls. Increased plasma THCy level was found in 10 children and adolescents from 12 cases homozygous for the C677T polymorphism of the MTHFR gene. No similar high frequency was observed in heterozygous subjects. Elevated fasting plasma TBARS levels were found in 38 cases and in 8 controls. The frequency differences were significant (p < 0.01). Allele frequency of the MTHFR polymorphism among cases and controls was similar. Significant correlation (r = 0.53, p < 0.02) was detected between plasma THCy and TBARS levels. One child had high serum TC level, 5 had low serum HDL-C level and all other children had normal serum TC, LDL-C, HDL-C and TG levels from children with hyperhomocysteinemia and/or high plasma TBARS levels. A significant correlation (r = 0.64, p < 0.01) was observed between plasma THCy levels of parents and children in the case group.Conclusion: The measurement of plasma THCy and TBARS levels may contribute to the detection of the risk of children and adolescents with high CHD risk family history.