Detection of polymorphisms of the mtDNA control region of Caretta caretta (Testudines: Cheloniidae) by PCR-SSCP

Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra-and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.

Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly in patients with heart failure

Background -: Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly and Thr164Ile were suggested to have an effect in heart failure. We evaluated these polymorphisms relative to clinical characteristics and prognosis of alarge cohort of patients with heart failure of different etiologies. Methods -: We studied 501 patients with heart failure of different etiologies. Mean age was 58 years (standard deviation 14.4 years), 298 (60%) were men. Polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism. Results -: During the mean follow-up of 12.6 months (standard deviation 10.3 months), 188 (38%) patients died. Distribution of genotypes of polymorphism Arg16Gly was different relative to body mass index (chi(2) = 9.797; p = 0.04). Overall the probability of survival was not significantly predicted by genotypes of Gln27Glu, Arg16Gly, or Thr164Ile. Allele and haplotype analysis also did not disclose any significant difference regarding mortality. Exploratory analysis through classification trees pointed towards a potential association between the Gln27Glu polymorphism and mortality in older individuals. Conclusion -: In this study sample, we were not able to demonstrate an overall influence of polymorphisms Gln27Glu and Arg16Gly of beta-2 receptor gene on prognosis. Nevertheless, Gln27Glu polymorphism may have a potential predictive value in older individuals.

Interaction between polymorphisms of the Human Leukocyte Antigen and HPV-16 Variants on the risk of invasive cervical cancer

Background: Persistent infection with oncogenic types of human papillomavirus (HPV) is the major risk factor for invasive cervical cancer (ICC), and non-European variants of HPV-16 are associated with an increased risk of persistence and ICC. HLA class II polymorphisms are also associated with genetic susceptibility to ICC. Our aim is to verify if these associations are influenced by HPV-16 variability. Methods: We characterized HPV-16 variants by PCR in 107 ICC cases, which were typed for HLA-DQA1, DRB1 and DQB1 genes and compared to 257 controls. We measured the magnitude of associations by logistic regression analysis. Results: European ( E), Asian-American ( AA) and African (Af) variants were identified. Here we show that inverse association between DQB1*05 ( adjusted odds ratio [ OR] = 0.66; 95% confidence interval [CI]: 0.39-1.12]) and HPV-16 positive ICC in our previous report was mostly attributable to AA variant carriers ( OR = 0.27; 95% CI: 0.10-0.75). We observed similar proportions of HLA DRB1*1302 carriers in E-P positive cases and controls, but interestingly, this allele was not found in AA cases ( p = 0.03, Fisher exact test). A positive association with DRB1*15 was observed in both groups of women harboring either E ( OR = 2.99; 95% CI: 1.13-7.86) or AA variants ( OR = 2.34; 95% CI: 1.00-5.46). There was an inverse association between DRB1*04 and ICC among women with HPV-16 carrying the 350T [83L] single nucleotide polymorphism in the E6 gene ( OR = 0.27; 95% CI: 0.08-0.96). An inverse association between DQB1*05 and cases carrying 350G (83V) variants was also found ( OR = 0.37; 95% CI: 0.15-0.89). Conclusion: Our results suggest that the association between HLA polymorphism and risk of ICC might be influenced by the distribution of HPV-16 variants.

Microsatellite Polymorphisms in Cassava Landraces from the Cerrado Biome, Mato Grosso do Sul, Brazil

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region`s high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.

Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.

Endothelial nitric oxide synthase polymorphisms and adaptation of parasympathetic modulation to exercise training

SILVA, B. M., F. J. NEVES, M. V. NEGRÃO, C. R. ALVES, R. G. DIAS, G. B. ALVES, A. C. PEREIRA, M. Urbana A. RONDON, J. E. KRIEGER, C. E. NEGRÃO, and A. C. DA NOBREGA. Endothelial Nitric Oxide Synthase Polymorphisms and Adaptation of Parasympathetic Modulation to Exercise Training. Med. Sci. Sports Exerc., Vol. 43, No. 9, pp. 1611-1618, 2011. Purpose: There is a large interindividual variation in the parasympathetic adaptation induced by aerobic exercise training, which may be partially attributed to genetic polymorphisms. Therefore, we investigated the association among three polymorphisms in the endothelial nitric oxide gene (-786T>C, 4b4a, and 894G>T), analyzed individually and as haplotypes, and the parasympathetic adaptation induced by exercise training. Methods: Eighty healthy males, age 20-35 yr, were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis, and haplotypes were inferred using the software PHASE 2.1. Autonomic modulation (i.e., HR variability and spontaneous baroreflex sensitivity) and peak oxygen consumption ((V) over dotO(2peak)) were measured before and after training (running, moderate to severe intensity, three times per week, 60 min.day(-1), during 18 wk). Results: Training increased (V) over dotO(2peak) (P < 0.05) and decreased mean arterial pressure (P < 0.05) in the whole sample. Subjects with the -786C polymorphic allele had a significant reduction in baroreflex sensitivity after training (change: wild type (-786TT) = 2% +/- 89% vs polymorphic (-786TC/CC) = -28% +/- 60%, median +/- quartile range, P = 0.03), and parasympathetic modulation was marginally reduced in subjects with the 894T polymorphic allele (change: wild type (894GG) = 8% +/- 67% vs polymorphic (894GT/TT) = -18% +/- 59%, median +/- quartile range, P = 0.06). Furthermore, parasympathetic modulation percent change was different between the haplotypes containing wild-type alleles(-786T/4b/894G) and polymorphic alleles at positions -786 and 894 (-786C/4b/894T) (-6% +/- 56% vs -41% +/- 50%, median T quartile range, P = 0.04). Conclusions: The polymorphic allele at position -786 and the haplotype containing polymorphic alleles at positions -786 and 894 in the endothelial nitric oxide gene were associated with decreased parasympathetic modulation after exercise training.

Influence of angiotensinogen and angiotensin-converting enzyme polymorphisms on cardiac hypertrophy and improvement on maximal aerobic capacity caused by exercise training

Background The allele threonine (T) of the angiotensinogen has been associated with ventricular hypertrophy in hypertensive patients and soccer players. However, the long-term effect of physical exercise in healthy athletes carrying the T allele remains unknown. We investigated the influence of methionine M or T allele of the angiotensinogen and D or I allele of the angiotensin-converting enzyme on left-ventricular mass index (LVMI) and maximal aerobic capacity in young healthy individuals after long-term physical exercise training. Design Prospective clinical trial. Methods Eighty-three policemen aged between 20 and 35 years (mean +/- SD 26 +/- 4.5 years) were genotyped for the M235T gene angiotensinogen polymorphism (TT, n=25; MM/MT, n=58) and angiotensin-converting enzyme gene insertion/deletion (I/D) polymorphism (11, n=18; DD/DI, n=65). Left-ventricular morphology was evaluated by echocardiography and maximal aerobic capacity (VO(2peak)) by cardiopulmonary exercise test before and after 17 weeks of exercise training (50-80% VO(2peak)). Results Baseline VO(2peak) and LVMI were similar between TT and MM/MT groups, and II and DD/DI groups. Exercise training increased significantly and similarly VO(2peak) in homozygous TT and MM/MT individuals, and homozygous II and DD/DI individuals. In addition, exercise training increased significantly LVMI in TT and MM/MT individuals (76.5 +/- 3 vs. 86.7 +/- 4, P=0.00001 and 76.2 +/- 2 vs. 81.4 +/- 2, P=0.00001, respectively), and II and DD/DI individuals (777 +/- 4 vs. 81.5 +/- 4, P=0.0001 and 76 +/- 2 vs. 83.5 +/- 2, P=0.0001, respectively). However, LVMI I in TT individuals was significantly greater than in MM/MT individuals (P=0.04). LVMI was not different between 11 and DD/DI individuals. Conclusion Left-ventricular hypertrophy caused by exercise training is exacerbated in homozygous TT individuals with angiotensinogen polymorphism. Eur J Cardiovasc Prev Rehabil 16:487-492 (C) 2009 The European Society of Cardiology

Genetic variability among sugarcane genotypes based on polymorphisms in sucrose metabolism and drought tolerance genes

Target region amplification polymorphism (TRAP) markers were used to estimate the genetic similarity (GS) among 53 sugarcane varieties and five species of the Saccharum complex. Seven fixed primers designed from candidate genes involved in sucrose metabolism and three from those involved in drought response metabolism were used in combination with three arbitrary primers. The clustering of the genotypes for sucrose metabolism and drought response were similar, but the GS based on Jaccard`s coefficient changed. The GS based on polymorphism in sucrose genes estimated in a set of 46 Brazilian varieties, all of which belong to the three Brazilian breeding programs, ranged from 0.52 to 0.9, and that based on drought data ranged from 0.44 to 0.95. The results suggest that genetic variability in the evaluated genes was lower in the sucrose metabolism genes than in the drought response metabolism ones.

Relationships between gene polymorphisms of folate-related proteins and vitamins and metabolites in pregnant women and neonates

Background: The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII) and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their neonates. Methods: Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates. MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP. Results: Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by combined genotypes MTHFR 677TT/GCPII 1561CC and MTHFR 677TT/RFC1 80AG. The MTHFR and RFC1 polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1 80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl, respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and by neonatal Cbl. Conclusions: Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism influenced tHcy in neonates. (C) 2008 Elsevier B.V. All rights reserved.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women

Objectives: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/ S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. Subjects/ Methods: Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism ( RFLP). Results: Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. Conclusions: Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

No Association Between MTHFR A1298C and MTRR A66G Polymorphisms, and MS in an Australian Cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ^2 = 0.005, P = 0.95, MTHFR χ^2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS.

Gene and genotypic diversity of Phytophthora cinnamomi in South Africa and Australia revealed by DNA polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D-m = 0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D-m South Africa = 0.020 and D-m Australia = 0.025 respectively), negative fixation indices, and significant deviations from Hardy-Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.

Association of tumor necrosis factor-alpha polymorphisms and ozone-induced change in lung function

Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor-alpha (TNF), lymphotoxin-alpha (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV1 with ozone challenge, as a percentage of baseline, was -3% in TNF -308G/A or A/A individuals, compared with -9% in G/G individuals (p = 0.024). When considering TNF haplotypes, the smallest change in FEV1 with ozone exposure was associated with the TNF haplotype comprising LTA +252G/TNF -1031T/TNF -308A/TNF -238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p = 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozone-induced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.