Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

Distribution of hepatitis c virus (hcv) genotypes in patients with chronic infection from Rondonia, Brazil

Background: Hepatitis C virus (HCV) is an important human pathogen affecting around 3% of the human population. In Brazil, it is estimated that there are approximately 2 to 3 million HCV chronic carriers. There are few reports of HCV prevalence in Rondonia State (RO), but it was estimated in 9.7% from 1999 to 2005. The aim of this study was to characterize HCV genotypes in 58 chronic HCV infected patients from Porto Velho, Rondonia (RO), Brazil. Methods: A fragment of 380 bp of NS5B region was amplified by nested PCR for genotyping analysis. Viral sequences were characterized by phylogenetic analysis using reference sequences obtained from the GenBank (n = 173). Sequences were aligned using Muscle software and edited in the SE-AL software. Phylogenetic analyses were conducted using Bayesian Markov chain Monte Carlo simulation (MCMC) to obtain the MCC tree using BEAST v. 1.5.3. Results: From 58 anti-HCV positive samples, 22 were positive to the NS5B fragment and successfully sequenced. Genotype 1b was the most prevalent in this population (50%), followed by 1a (27.2%), 2b (13.6%) and 3a (9.0%). Conclusions: This study is the first report of HCV genotypes from Rondonia State and subtype 1b was found to be the most prevalent. This subtype is mostly found among people who have a previous history of blood transfusion but more detailed studies with a larger number of patients are necessary to understand the HCV dynamics in the population of Rondonia State, Brazil.

T allele of-344C/T polymorphism in aldosterone synthase gene is not associated with resistant hypertension

Resistant hypertension (RH) is the maintenance of elevated blood pressure concurrent with the use of three different antihypertensive drugs, one of which is a diuretic. The Renin-Angiotensin-Aldosterone System plays a major role in volume-dependent hypertension. Therefore, its components are interesting targets for genetic association studies. This work focused on the -344 C/T polymorphism in the CYP11b2 gene, which encodes aldosterone synthase. This work evaluates the association between T allele and resistance to anti-hypertensive treatment. Genotyping analysis included 88 subjects with RH, 142 who were responsive to anti-hypertensive treatment and 110 subjects as a control group. Plasmatic concentrations of aldosterone, renin and cortisol, carotid intima-media thickness and carotid-femoral pulse wave velocity were assessed in a smaller subset of hypertensive patients. An association was found between T allele and hypertension (P < 0.005), but there was no difference in allele frequencies between both hypertensive groups. There was no difference in plasmatic parameters either, in remodeling indicators between the genotypic groups.

Prevalence and genotypes of hepatitis C virus among injecting drug users from Salvador-BA, Brazil

Hepatitis C virus (HCV) is the major infectious disease agent among injecting drug users (IDUs), with seroprevalence ranging from 50-90%. In this paper, serological and virological parameters were investigated among 194 IDUs, 94 ex-IDUs and 95 non-IDUs that were sampled by the "snowball" technique in three localities renowned for both intense drug use and trafficking activities in Salvador, Brazil. The majority of the participants were male, but sex and mean age differed significantly between IDUs/ex-IDUs and non-IDUs (p < 0.05). Anti-HCV screening revealed that 35.6%, 29.8% and 5.3% of samples from IDUs, ex-IDUs and non-IDUs, respectively, were seropositive. HCV-RNA detection confirmed that the prevalence of infection was 29.4%, 21.3% and 5.3% for IDUs, ex-IDUs and non-IDUs, respectively. Genotyping analysis among IDUs/ex-IDUs determined that 76.9% were infected with genotype 1, 18.5% with genotype 3 and 4.6% with a mixed genotype; this result differed significantly from non-IDUs, where genotype 3 was the most frequent (60%), followed by genotype 1 (20%) and a mixed genotype (20%). We report a significantly higher prevalence of HCV infection in IDUs/ex-IDUs compared to the control group (p < 0.001). Although the sample size of our study was small, the differences in HCV genotype distribution reported herein for IDUs/ex-IDUs and non-IDUs warrant further investigation.

Reduced folate carrier 1 (RFC1) is associated with cleft of the lip only

In this report, we have reanalyzed genotyping data in a collection of families from South America based on maternal origin. Genotyping analysis was performed at the Craniofacial Anomalies Research Center at the University of Iowa. These genotypes were derived from genomic DNA samples obtained from blood spots from children born with isolated orofacial clefts in 45 hospitals located in eight countries (Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela) collaborating with ECLAMC (Latin American Collaborative Studies of Congenital Malformations) between January 1998 and December 1999. Dried blood samples were sent by regular mail to the Laboratory of Congenital Malformations, Federal University of Rio de Janeiro. Previous findings suggested that mitochondrial haplotype D is more commonly found among cleft cases born in South America. We hypothesized that association of certain genes may depend upon the ethnic origin, as defined by population-specific markers. Therefore, we tested if markers in MTHFR (5,10-methylenetetrahydrofolate reductase) and RFC1 (reduced folate carrier 1) were associated with oral clefts, depending on the maternal origin defined by the mitochondrial haplotype. Transmission distortion of alleles in MTHFR C677T and RFC1 G80A polymorphic variants was tested in 200 mother/affected child pairs taking into consideration maternal origin. RFC1 variation was over-transmitted to children born with cleft lip only (P = 0.017) carrying mitochondrial DNA haplotypes other than haplotype D. Our results provide a new indication that variation in RFC1 may contribute to cleft lip only. Future studies should investigate the association between oral clefts and RFC1 based on more discrete phenotypes.

Analyse génotypique des cellules initiatrices de tumeurs exprimant CD133 dans le neuroblastome

Le neuroblastome (NB) est la tumeur solide extracranienne la plus fréquente et mortelle chez les jeunes enfants. Il se caractérise par une résistance à la chimiothérapie possiblement en partie dû à la présence de cellules initiatrices de tumeurs (TICs). Des études ont mis en évidence le rôle de CD133 comme un marqueur des TICs dans divers types de cancers. Les buts de notre travail étaient d’abord de démontrer les vertus de TICs des cellules exprimant CD133 et ensuite, en utilisant une analyse globale du génome avec des polymorphismes nucléotidiques simples (SNPs), d’effectuer une analyse différentielle entre les TICs et les autres cellules du NB afin d’en identifier les anomalies génétiques spécifiques. Des lignées cellulaires de NB ont été triées par cytométrie de flux afin d’obtenir deux populations: une enrichie en CD133 (CD133high), l’autre faible en CD133 (CD133low). Afin de déterminer si ces populations cellulaires présentent des propriétés de TICs, des essais sur les neurosphères, les colonies en agar mou et les injections orthotopiques de 500 cellules sélectionnées dans 11 souris ont été réalisées. Après une isolation de l’ADN des populations sélectionnées, nous avons effectué une analyse génotypique par SNP utilisant les puces « Affymetrix Genome-Wide Human SNP Array 6.0 ». Pour vérifier l’expression des gènes identifiés, des Western Blots ont été réalisés. Nos résultats ont démontré que la population CD133 avait des propriétés de TICs in vitro et in vivo. L’analyse génotypique différentielle a permis d’identifier deux régions communes (16p13.3 and 19p13.3) dans la population CD133high ayant des gains et deux autres régions (16q12.1 and 21q21.3) dans la population CD133low possédant des pertes d’hétérozygoties (LOH). Aucune perte n’a été observée. Parmi les gènes étudiés, l’expression protéique d’éphrine-A2 était corrélée à celle de CD133 dans 6 tumeurs et 2 lignées cellulaires de NB. De plus, l’augmentation de la concentration d’anticorps anti-éphrine-A2 dans le milieu diminue la taille des neurosphères. Ainsi, la population CD133high, qui a des vertus de TICs, possède des caractéristiques génotypiques différentes par rapport à celle CD133low. La présence d’éphrine-A2 dans les cellules exprimant CD133 souligne son importance dans le développement des TICs. Ces résultats suggèrent la présence de potentielle cible pour de nouvelles thérapeutiques ciblant les TICs mise en évidence par l’étude génomique.

Caractérisation génétique d’une forme d’ataxie tardive.

Les ataxies forment un groupe de maladies neurodégénératives qui sont caractérisées par un manque de coordination des mouvements volontaires. Mes travaux ont porté sur une forme d'ataxie à début tardif (LOCA), après l’âge de 50 ans. Les principales caractéristiques cliniques sont: atrophie cérébelleuse à l’IRM (88%), dysarthrie (81%), atrophie du lobe frontal (50%) et nystagmus (52%). La ségrégation dans les familles de cette ataxie est en faveur d’une transmission récessive. Afin d'identifier le gène responsable de LOCA, nous avons recruté 38 patients affectés d'une forme tardive d'ataxie, issus du SLSJ, des Cantons de l’Est ou d’autres régions du Québec. Un premier criblage du génome a été effectué avec des marqueurs microsatellites sur une famille clé. Une analyse de liaison paramétrique nous a suggéré une liaison au chromosome 13 (4.4Mb). Une recherche d’un haplotype partagé entre 17 familles LOCA a diminué la taille de l'intervalle candidat à 1.6Mb, mais l’haplotype s’est avéré fréquent dans la population canadienne-française. Un second criblage du génome avec des marqueurs SNP nous a permis d’évaluer par cartographie d’homozygotie la possibilité qu’une mutation fondatrice partagée dans des sous-groupes de malades. Plusieurs stratégies d'analyse ont été effectuées, entre autre par regroupement régional. Aucun loci candidats ne fut identifié avec confiance. Nous avons donc combiné les données de génotypage avec le séquençage exomique afin d'identifier le gène responsable. L'analyse de six individus atteints nous a permis d'obtenir une liste de variants rare contenant quatre gènes potentiels. Cette analyse doit se poursuivre pour identifier le gène responsable de LOCA.

Burst oxidativo dos neutrófilos humanos: estudo da influência do polimorfismo do receptor para IgG FeyRIIIb na cooperação com os receptores para complemento

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Distribution of hepatitis c virus (hcv) genotypes in patients with chronic infection from Rondônia, Brazil

Abstract Background Hepatitis C virus (HCV) is an important human pathogen affecting around 3% of the human population. In Brazil, it is estimated that there are approximately 2 to 3 million HCV chronic carriers. There are few reports of HCV prevalence in Rondônia State (RO), but it was estimated in 9.7% from 1999 to 2005. The aim of this study was to characterize HCV genotypes in 58 chronic HCV infected patients from Porto Velho, Rondônia (RO), Brazil. Methods A fragment of 380 bp of NS5B region was amplified by nested PCR for genotyping analysis. Viral sequences were characterized by phylogenetic analysis using reference sequences obtained from the GenBank (n = 173). Sequences were aligned using Muscle software and edited in the SE-AL software. Phylogenetic analyses were conducted using Bayesian Markov chain Monte Carlo simulation (MCMC) to obtain the MCC tree using BEAST v.1.5.3. Results From 58 anti-HCV positive samples, 22 were positive to the NS5B fragment and successfully sequenced. Genotype 1b was the most prevalent in this population (50%), followed by 1a (27.2%), 2b (13.6%) and 3a (9.0%). Conclusions This study is the first report of HCV genotypes from Rondônia State and subtype 1b was found to be the most prevalent. This subtype is mostly found among people who have a previous history of blood transfusion but more detailed studies with a larger number of patients are necessary to understand the HCV dynamics in the population of Rondônia State, Brazil.

A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.

Regulation of two P450 isoforms: CYP2D6 and CYP4F11

Cytochromes P450 catalyze a monooxygenase reaction in which molecular oxygen is split and one oxygen atom is incorporated into the substrate. As a whole, P450 researchers have focused most of their attention on substrate metabolism and relatively little on how these enzymes are regulated. This study will focus on the regulation of two P450 isoforms known as, CYP2D6 and CYP4F11. ^ The human CYP2D gene locus contains two pseudogenes and one functional gene known as CYP2D6. This locus is highly polymorphic and produces several alternatively spliced transcripts from the pseudogene CYP2D7. My objective was to understand the role of SV5-in (splice variant 5), one of several alternative splice variants transcribed from the CYP2D7 pseudogene. My results indicate that SV5-in mRNA causes an increase in CYP2D6 protein levels and suggest that there is a role for SV5-in in regulation of CYP2D6 expression. ^ Second, CYP4F11 is a recently discovered and uncharacterized isoform, derived from the CYP4F subfamily. It metabolizes several clinically relevant drugs (i.e.—erythromycin and benzphetamine) and some endogenous inflammatory mediators (i.e.—LTB4). After evaluation of microarray data, I observed an increase in CYP4F11 mRNA levels from wild-type HCT116 cells compared to p53-null cells. Our objectives were to explore and understand this connection between p53 and CYP4F11. Microarray data were confirmed by Q-PCR, after which this effect was again observed at the protein level via Western blot and again at the promoter level via luciferase assay and chromatin immunoprecipitation. Our results indicate that p53 protein regulates expression of CYP4F11 mRNA and protein through CYP4F11 promoter binding (note that p53 binding to CYP4F11 DNA was not shown to be direct). These results signify a whole new level of regulation of drug metabolizing enzymes by p53. ^ An understanding of CYP4F11 regulation by p53 could help us understand another pathway leading to apoptosis or cell growth arrest. This can aid future drug studies and discover new drug metabolism pathways under the control of a tumor suppressor protein. An understanding of the CYP2D6 regulation pathway could illuminate the role of non-coding RNAs in the P450 field and potentially explain several inter-individual drug response variations observed in clinical medicine that are not yet completely explained by genotyping analysis. ^

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Analysis of DNA polymorphism in ancient barley herbarium material: validation of the KASP SNP genotyping platform

The availability of crop specimens archived in herbaria and old seed collections represent valuable resources for the analysis of plant genetic diversity and crop domestication. The ability to extract ancient DNA (aDNA) from such samples has recently allowed molecular genetic investigations to be undertaken in ancient materials. While analyses of aDNA initially focused on the use of markers which occur in multiple copies such as the internal transcribed spacer region (ITS) within ribosomal DNA and those requiring amplification of short DNA regions of variable length such as simple sequence repeats (SSRs), emphasis is now moving towards the genotyping of single nucleotide polymorphisms (SNPs), traditionally undertaken in aDNA by Sanger sequencing. Here, using a panel of barley aDNA samples previously surveyed by Sanger sequencing for putative causative SNPs within the flowering-time gene PPD-H1, we assess the utility of the Kompetitive Allele Specific PCR (KASP) genotyping platform for aDNA analysis. We find KASP to out-perform Sanger sequencing in the genotyping of aDNA samples (78% versus 61% success, respectively), as well as being robust to contamination. The small template size (≥46 bp) and one-step, closed-tube amplification/genotyping process make this platform ideally suited to the genotypic analysis of aDNA, a process which is often hampered by template DNA degradation and sample cross-contamination. Such attributes, as well as its flexibility of use and relatively low cost, make KASP particularly relevant to the genetic analysis of aDNA samples. Furthermore, KASP provides a common platform for the genotyping and analysis of corresponding SNPs in ancient, landrace and modern plant materials. The extended haplotype analysis of PPD-H1 undertaken here (allelic variation at which is thought to be important for the spread of domestication and local adaptation) provides further resolution to the previously identified geographic cline of flowering-time allele distribution, illustrating how KASP can be used to aid genetic analyses of aDNA from plant species. We further demonstrate the utility of KASP by genotyping ten additional genetic markers diagnostic for morphological traits in barley, shedding light on the phenotypic traits, alleles and allele combinations present in these unviable ancient specimens, as well as their geographic distributions.

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of similar to 1.7 kb located in 2 chromosomal bands of 600-720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes. (c) 2010 Elsevier Ireland Ltd. All rights reserved.