EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of echinococcus multilocularis

Alveolar echinococcosis (AE)--caused by the cestode Echinococcus multilocularis--is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

Parentage analysis with genetic markers in natural populations. I. The expected proportion of offspring with unambiguous paternity.

Recent studies indicate that polymorphic genetic markers are potentially helpful in resolving genealogical relationships among individuals in a natural population. Genetic data provide opportunities for paternity exclusion when genotypic incompatibilities are observed among individuals, and the present investigation examines the resolving power of genetic markers in unambiguous positive determination of paternity. Under the assumption that the mother for each offspring in a population is unambiguously known, an analytical expression for the fraction of males excluded from paternity is derived for the case where males and females may be derived from two different gene pools. This theoretical formulation can also be used to predict the fraction of births for each of which all but one male can be excluded from paternity. We show that even when the average probability of exclusion approaches unity, a substantial fraction of births yield equivocal mother-father-offspring determinations. The number of loci needed to increase the frequency of unambiguous determinations to a high level is beyond the scope of current electrophoretic studies in most species. Applications of this theory to electrophoretic data on Chamaelirium luteum (L.) shows that in 2255 offspring derived from 273 males and 70 females, only 57 triplets could be unequivocally determined with eight polymorphic protein loci, even though the average combined exclusionary power of these loci was 73%. The distribution of potentially compatible male parents, based on multilocus genotypes, was reasonably well predicted from the allele frequency data available for these loci. We demonstrate that genetic paternity analysis in natural populations cannot be reliably based on exclusionary principles alone. In order to measure the reproductive contributions of individuals in natural populations, more elaborate likelihood principles must be deployed.

Neutral behavior of shared polymorphism

Several cases have been described in the literature where genetic polymorphism appears to be shared between a pair of species. Here we examine the distribution of times to random loss of shared polymorphism in the context of the neutral Wright–Fisher model. Order statistics are used to obtain the distribution of times to loss of a shared polymorphism based on Kimura’s solution to the diffusion approximation of the Wright–Fisher model. In a single species, the expected absorption time for a neutral allele having an initial allele frequency of ½ is 2.77 N generations. If two species initially share a polymorphism, that shared polymorphism is lost as soon as either of two species undergoes fixation. The loss of a shared polymorphism thus occurs sooner than loss of polymorphism in a single species and has an expected time of 1.7 N generations. Molecular sequences of genes with shared polymorphism may be characterized by the count of the number of sites that segregate in both species for the same nucleotides (or amino acids). The distribution of the expected numbers of these shared polymorphic sites also is obtained. Shared polymorphism appears to be more likely at genetic loci that have an unusually large number of segregating alleles, and the neutral coalescent proves to be very useful in determining the probability of shared allelic lineages expected by chance. These results are related to examples of shared polymorphism in the literature.

Case-only study of interactions between genetic polymorphisms of GSTM1, P1, T1 and Z1 and smoking in Parkinson's disease

Current opinion contends that complex interactions between genetic and environmental factors play a role in the etiology of Parkinson's disease (PD). Cigarette smoking is thought to reduce risk of PD, and emerging evidence suggests that genetic factors may modulate smoking's effect. We used a case-only design, an approach not previously used to study gene-environment interactions in PD, specifically to study interactions between glutathione-S-transferase (GST) gene polymorphisms and smoking in relation to PD. Four-hundred PD cases (age at onset: 60.0 +/- 10.7 years) were genotyped for common polymorphisms in GSTM1, PI, T1 and Z1 using well-established methods. Smoking exposure data were collected in face-to-face interviews. The independence of the studied GST genotypes and smoking exposure was confirmed by studying 402 healthy, aged individuals. No differences were observed in the distributions of GSTM1, T1 or Z1 polymorphisms between ever-smoked and never-smoked PD cases using logistic regression (all P > 0.43). However, GSTP1 *C haplotypes were over-represented among PD cases who ever smoked (odds ratio for interaction (ORi) = 2.00 (95% Cl: 1.11-3.60, P = 0.03)). Analysis revealed that ORi between smoking and the GSTP1-114Val carrier status increased with increasing smoking dose (P = 0.02 for trend). These data suggest that one or more GSTP1 polymorphisms may interact with cigarette smoking to influence the risk for PD. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Meta-analysis of the population and phenotypic expression of CYP2C9/2C19 polymorphism on drug metabolism in different ethnicities

The term "pharmacogenetics" has been defined as the scientific study of inherited factors that affect the human drug response. Many pharmacogenetie studies have been published since 1995 and have focussed on the principal enzyme family involved in drug metabolism, the cytochrome P450 family, particularly cytochrome P4502C9 and 2C19. In order to investigate the pharmacogenetic aspect of pharmacotherapy, the relevant studies describing the association of pharmacogenetic factor(s) in drug responses must be retrieved from existing literature using a systematic review approach. In addition, the estimation of variant allele prevalence for the gene under study between different ethnic populations is important for pharmacogenetic studies. In this thesis, the prevalence of CYP2C9/2C19 alleles between different ethnicities has been estimated through meta-analysis and the population genetic principle. The clinical outcome of CYP2C9/2C19 allelic variation on the pharmacotherapy of epilepsy has been investigated; although many new antiepileptic drugs have been launched into the market, carbamazepine, phenobarbital and phenytoin are still the major agents in the pharmacotherapy of epilepsy. Therefore, phenytoin was chosen as a model AED and the effect of CYP2C9/2C19 genetic polymorphism on phenytoin metabolism was further examined.An estimation of the allele prevalence was undertaken for three CYP2C9/2C19 alleles respectively using a meta-analysis of studies that fit the Hardy-Weinberg equilibrium. The prevalence of CYP2C9*1 is approximately 81%, 96%, 97% and 94% in Caucasian, Chinese, Japanese, African populations respectively; the pooled prevalence of CYP2C19*1 is about 86%, 57%, 58% and 85% in these ethnic populations respectively. However, the studies of association between CYP2C9/2C19 polymorphism and phenytoin metabolism failed to achieve any qualitative or quantitative conclusion. Therefore, mephenytoin metabolism was examined as a probe drug for association between CYP2C19 polymorphism and mephenytoin metabolic ratio. Similarly, analysis of association between CYP2C9 polymorphism and warfarin dose requirement was undertaken.It was confirmed that subjects carrying two mutated CYP2C19 alleles have higher S/R mephenytoin ratio due to deficient CYP2C19 enzyme activity. The studies of warfarin and CYP2C9 polymorphism did not provide a conclusive result due to poor comparability between studies.The genetic polymorphism of drug metabolism enzymes has been studied extensively, however other genetic factors, such as multiple drug resistance genes (MDR) and genes encoding ion channels, which may contribute to variability in function of drug transporters and targets, require more attention in future pharmacogenetic studies of antiepileptic drugs.

Interação entre consumo alimentar e polimorfismos da GSTM1 e GSTT1 no risco para o câncer de cabeça e pescoço: estudo caso-controle em São Paulo, Brasil

O objetivo foi investigar a interação entre fatores dietéticos e polimorfismos de enzimas de metabolização de xenobióticos (GSTM1 e GSTT1) associadas ao câncer de cabeça e pescoço em um estudo caso controle de base hospitalar, no Município de São Paulo, Brasil. Participaram 103 casos incidentes, histologicamente confirmados, e 101 controles. O consumo alimentar foi obtido por um questionário de frequência alimentar validado. Os polimorfismos GSTM1 e GSTT1 foram avaliados pelo método PCR. Observou-se aumento de risco no mais alto tercil de consumo de carne bovina na presença do alelo nulo da GSTM1 (OR = 10,79; IC95%: 2,17-53,64) e GSTT1 (OR = 3,41; IC95%: 0,43-27,21). Considerando-se a razão entre alimentos de origem animal e vegetal, verificou-se para o tercil intermediário a OR = 2,02 (IC95%: 0,24-16,0) e no tercil superior OR = 3,23 (IC95%: 0,40-25,92). Os resultados apontam para uma possível interação entre o consumo de carne e variantes polimórficas dos genes GSTM1 e GSTT1 na modulação do risco para o câncer de cabeça e pescoço, influenciados pelo consumo de alimentos de origem vegetal.

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*001 was the most common allele found, followed by ABO*022 and by ABO*A103. We identified 22 new ABO*(variants) in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid

Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that catalyses the acetylation of arylamine and hydrazine drugs as well as several known carcinogens, and so its levels in the body may have toxicological importance with regard to drug toxicity and cancer risk. Recently, we showed that p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultured cells, but the exact mechanism by which PABA acts remains unclear. In the present study, we investigated the possibility that PABA-induced down-regulation involves its metabolism to N-OH-PABA, since N-OH-AAF functions as an irreversible inhibitor of hamster and rat NAT1. We show here that N-OH-PABA irreversibly inactivates human NAT1 both in cultured cells and cell cytosols in a time- and concentration-dependent manner. Maximal inactivation in cultured cells occurred within 4 hr of treatment, with a concentration of 30 muM reducing activity by 60 +/- 7%. Dialysis studies showed that inactivation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement of the cofactor-binding site. In agreement with these data, kinetic studies revealed a 4-fold increase in cofactor K-m, but no change in substrate K-m for N-OH-PABA-treated cytosols compared to control. We conclude that N-OH-PABA decreases NAT1 activity by a direct interaction with the enzyme and appears to be a result of covalent modification at the cofactor-binding site. This is in contrast to our findings for PABA, which appears to reduce NAT1 activity by down-regulating the enzyme, leading to a decrease in NAT1 protein content. BIOCHEM PHARMACOL 60;12: 1829-1836, 2000. (C) 2000 Elsevier Science Inc.

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in Aborigines from Western Australia

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes, Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed, The wild-type allele *1 was the most frequent (85.8%) and the non-functional alleles (*4, *5, *16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5, For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype, For CYP2E1, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6*10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively, Pharmacogenetics 11:69-76 (C) 2001 Lippincott Williams & Wilkins.

Maternal and neonatal interleukin-1 receptor antagonist genotype and pregnancy outcome in a population with a high rate of pre-term birth

Problem We evaluated associations between a length polymorphism in intron 2 of the gene coding for IL-1ra (gene symbol IL1RN) and pregnancy outcome in a population with a high rate of preterm birth. Method of study Subjects were pregnant women in Maceio, Brazil and their newborns. DNA was tested for IL1RN genotypes and alleles by gene amplification using primer pairs that spanned the polymorphic region. Every subject completed a detailed questionnaire. Results The frequency of allele 2 (IL1RN*2) carriage was elevated in mothers with a spontaneous preterm birth (SPTB) in the current pregnancy (P = 0.02) and also with a prior preterm delivery (P = .01). Both SPTB with intact membranes (P = 0.01) and SPTB preceded by pre-term pre-mature rupture of membranes (P = .03) were associated with IL1RN*2 carriage. A previous fetal demise was more than twice as prevalent in mothers positive for two copies of IL1RN*2. Conclusion Maternal carriage of IL1RN*2 increases susceptibility to inflammation-triggered spontaneous pre-term birth.

Metabolic activation of aromatic amines by human pancreas

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas, Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens, Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethylamine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/ 4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [H-3]N-hydroxy-ABP) of pancreatic cytosols was high, about two-thirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using P-32-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.

Hypotension caused by therapeutic doses of venlafaxine: case report and proposed pathophysiological mechanisms

Although venlafaxine is usually associated with modest increases in blood pressure and not so often clinical hypertension, there are a few reported cases of hypotension related to overdoses of this specific antidepressant. The case study of a young female patient with a history of Major Depressive Disorder who initiated treatment with venlafaxine 75 mg/day and developed hypotension when the dosage was titrated up to 225 mg/day is described. The patient did not present comorbid diseases nor use other medication. A temporal association and a dose-dependent relationship between the hypotension and the use of venlafaxine is shown. To the best of the knowledge of the authors, this is the first case report that specifically associates regular doses of venlafaxine with the presence of hypotension. A pathophysiological mechanism is proposed, involving the participation of presynaptic alpha2-adrenergic receptors and the presence of a possible genetic polymorphism of cytochrome P4502D6, which is associated with lower drug metabolization, to explain the relationship between venlafaxine in regular dosage and development of hypotension.

DC-SIGN (CD209) gene promoter polymorphisms in a Brazilian population and their association with human T-cell lymphotropic virus type 1 infection

This study evaluated four polymorphisms located in the DC-SIGN (CD209) gene promoter region (positions -336, -332 -201 and -139) in DNA samples from four Brazilian ethnic groups (Caucasians, Afro-Brazilian, Asians and Amerindians) to establish the population distribution of these single-nucleotide polymorphisms (SNPs) and correlated DC-SIGN polymorphisms and infection in samples from human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals. To identify CD209 SNPs, 452 bp of the CD209 promoter region were sequenced and the genotype and allelic frequencies were evaluated. This is the first study to show genetic polymorphism in the CD209 gene in distinct Brazilian ethnic groups with the distribution of allelic and genotypic frequency. The results showed that -336A and -139A SNPs were quite common in Asians and that the -201T allele was not observed in Caucasians, Asians or Amerindians. No significant differences were observed between individuals with HTLV-1 disease and asymptomatic patients. However, the -336A variant was more frequent in HTLV-1 -infected patients [HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 80%; healthy asymptomatic HTLV-1 carriers, 90 %] than in the control group (70 %) [P=0.0197, odds ratio (OR)=2.511, 95 % confidence interval (CI)=1.218-5.179). In addition, the -139A allele was found to be associated with protection against HTLV-1 infection (P=0.0037, OR=0.3758, 95% CI=0.1954-0.7229) when the HTLV-1 -infected patients as a whole were compared with the healthy-control group. These observations suggest that the -139A allele may be associated with HTLV-1 infection, although no significant association was observed among asymptomatic and HAM/TSP patients. In conclusion, the variation observed in SNPs -336 and -139 indicates that this lectin may be of crucial importance in the susceptibility/transmission of HTLV-1 infections.