Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa.

PURPOSE: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. METHODS: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. RESULTS: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. CONCLUSIONS: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.

Prospective study of insulin-like growth factor-I, insulin-like growth factor-binding protein 3, genetic variants in the IGF1 and IGFBP3 genes and risk of coronary artery disease.

Although experimental studies have suggested that insulin-like growth factor I (IGF-I) and its binding protein IGFBP-3 might have a role in the aetiology of coronary artery disease (CAD), the relevance of circulating IGFs and their binding proteins in the development of CAD in human populations is unclear. We conducted a nested case-control study, with a mean follow-up of six years, within the EPIC-Norfolk cohort to assess the association between circulating levels of IGF-I and IGFBP-3 and risk of CAD in up to 1,013 cases and 2,055 controls matched for age, sex and study enrolment date. After adjustment for cardiovascular risk factors, we found no association between circulating levels of IGF-I or IGFBP-3 and risk of CAD (odds ratio: 0.98 (95% Cl 0.90-1.06) per 1 SD increase in circulating IGF-I; odds ratio: 1.02 (95% Cl 0.94-1.12) for IGFBP-3). We examined associations between tagging single nucleotide polymorphisms (tSNPs) at the IGF1 and IGFBP3 loci and circulating IGF-I and IGFBP-3 levels in up to 1,133 cases and 2,223 controls and identified three tSNPs (rs1520220, rs3730204, rs2132571) that showed independent association with either circulating IGF-I or IGFBP-3 levels. In an assessment of 31 SNPs spanning the IGF1 or IGFBP3 loci, none were associated with risk of CAD in a meta-analysis that included EPIC-Norfolk and eight additional studies comprising up to 9,319 cases and 19,964 controls. Our results indicate that IGF-I and IGFBP-3 are unlikely to be importantly involved in the aetiology of CAD in human populations.

Immunopathogenesis of Asthma and Atopic Diseases – The specific Role of a selected Panel of Genes in human T helper Cell Differentiation

T helper cell (Th) functions are crucial for proper immune defence against various intra- and extracellular pathogens. According to the specific immune responses, Th cells can be classified into subtypes, Th1 and Th2 cells being the most frequently characterized classes. Th1 and Th2 cells interact with other immune cells by regulating their functions with specific cytokine production. IFN, IL-2 and TNF- are the cytokines predominantly produced by Th1 cells whereas Th2 cells produce Th2-type cytokines, such as IL-4, IL-5 and IL-13. Upon TCR activation and in the presence of polarizing cytokines, Th cells differentiate into effector subtypes from a common precursor cell. IFN and IL-12 are the predominant Th1 polarizing cytokines whereas IL-4 directs Th2 polarization. The cytokines mediate their effects through specific receptor signalling. The differentiation process is complex, involving various signalling molecules and routes, as well as functions of the specific transcription factors. The functions of the Th1/Th2 cells are tightly regulated; however, knowledge on human Th cell differentiation is, as yet, fairly poor. The susceptibility for many immune-mediated disorders often originates from disturbed Th cell responses. Thus, research is needed for defining the molecular mechanisms involved in the differentiation and balanced functions of the Th cells. Importantly, the new information obtained will be crucial for a better understanding of the pathogenesis of immune-mediated disorders, such as asthma or autoimmune diseases. In the first subproject of this thesis, the role of genetic polymorphisms in the human STAT6, GATA3 and STAT4 genes were investigated for asthma or atopy susceptibility in Finnish asthma families by association analysis. These genes code for key transcription factors regulating Th cell differentiation. The study resulted in the identification of a GATA3 haplotype that associated with asthma and related traits (high serum IgE level). In the second subproject, an optimized method for human primary T cell transfection and enrichment was established. The method can be utilized for functional studies for the selected genes of interest. The method was also utilized in the third subproject, which aimed at the identification of novel genes involved in early human Th cell polarization (0-48h) using genome-wide oligonucleotide arrays. As a result, numerous genes and ESTs with known or unknown functions were identified in the study. Using an shRNA knockdown approach, a panel of novel IL-4/STAT6 regulated genes were identified in the functional studies of the genes. Moreover, one of the genes, NDFIP2, with a previously uncharacterized role in the human Th differentiation, was observed to promote IFN production of the differentiated Th1 cells. Taken together, the results obtained have revealed potential new relevant candidate genes serving as a basis for further studies characterizing the detailed networks involved in the human Th cell differentiation as well as in the genetic susceptibility of Th-mediated immune disorders.

Loss of egg yolk genes in mammals and the origin of lactation and placentation

Embryonic development in nonmammalian vertebrates depends entirely on nutritional reserves that are predominantly derived from vitellogenin proteins and stored in egg yolk. Mammals have evolved new resources, such as lactation and placentation, to nourish their developing and early offspring. However, the evolutionary timing and molecular events associated with this major phenotypic transition are not known. By means of sensitive comparative genomics analyses and evolutionary simulations, we here show that the three ancestral vitellogenin-encoding genes were progressively lost during mammalian evolution (until around 30-70 million years ago, Mya) in all but the egg-laying monotremes, which have retained a functional vitellogenin gene. Our analyses also provide evidence that the major milk resource genes, caseins, which have similar functional properties as vitellogenins, appeared in the common mammalian ancestor approximately 200-310 Mya. Together, our data are compatible with the hypothesis that the emergence of lactation in the common mammalian ancestor and the development of placentation in eutherian and marsupial mammals allowed for the gradual loss of yolk-dependent nourishment during mammalian evolution

Polymorphisms in candidate genes and their association with carcass traits and meat quality in Nellore cattle

The objective of this work was to estimate the allele polymorphism frequencies of genes in Nellore cattle and associate them with meat quality and carcass traits. Six hundred males were genotyped for the following polymorphisms: DGAT1 (VNTR with 18 nucleotides at the promoter region); ANK1, a new polymorphism, identified and mapped here at the gene regulatory region NW_001494427.3; TCAP (AY428575.1:g.346G>A); and MYOG (NW_001501985:g.511G>C). In the association study, phenotype data of hot carcass weight, ribeye area, backfat thickness, percentage of intramuscular fat, shear force, myofibrillar fragmentation index, meat color (L*, a*, b*), and cooking losses were used. Allele B from the ANK1 gene was associated with greater redness (a*). Alleles 5R, 6R, and 7R from the DGAT1 VNTR gene were associated with increased intramuscular fat, reduced cooking losses and increased ribeye area, respectively. The single nucleotide polymorphism (SNP) of the TCAP gene was not polymorphic, and MYOG alleles were not associated with any of the evaluated characteristics. These results indicate that ANK1 and DGAT1 genes can be used in the selection of Nellore cattle for carcass and meat quality.

Desempenho, características de carcaça e expressão de genes em tourinhos alimentados com lipídeos e monensina

O objetivo deste trabalho foi avaliar o desempenho, as características de carcaça e as suas correlações com as expressões gênicas em tourinhos. Quarenta animais Red Norte foram confinados e distribuídos em delineamento inteiramente casualizado, em arranjo fatorial 2x2, e alimentados com grãos de soja moídos ou gordura protegida, com ou sem inclusão de monensina. As dietas foram formuladas para serem isonitrogenadas, com teores de extrato etéreo semelhante (6,5%), e tiveram silagem de milho como volumoso (40%). O período experimental foi de 84 dias, precedido por 14 dias de adaptação. Foram avaliadas as expressões dos genes PPARA, SREBP1c e SCD1. Animais alimentados com gordura protegida tiveram maior ganho de peso nos primeiros 56 dias de confinamento. O uso da monensina não afetou o ganho de peso diário dos animais, mas proporcionou aumento do rendimento de carcaça. Houve correlação de -0,40 entre o ganho de peso dos animais e a expressão do SCD1. O uso de gordura protegida aumenta o desempenho dos animais no início do confinamento, e a monensina proporciona maior rendimento de carcaça.

Associação de SNPs nos genes para κ-caseína e β-lactoglobulina com curvas de lactação em cabras leiteiras

O objetivo deste trabalho foi identificar o modelo com melhor ajuste aos dados de produção de leite, gordura, proteína e sólidos totais para cabras leiteiras, bem como associar polimorfismos de nucleotídeo único (SNPs) nos genes para κ-caseína (κ-CSN3) e β-lactoglobulina (β-LG) aos parâmetros das curvas de produção e qualidade do leite. Foram avaliados 4.160 registros de produção de leite, gordura, proteína e sólidos totais de cabras das raças Alpina, Saanen e mestiça, no Estado de Antioquia, na Colômbia. Os modelos não lineares com melhor ajuste à estrutura dos dados foram os de Nelder, para produção de leite, e de Cappio-Borlino, para qualidade do leite. As análises de associação mostraram efeito significativo do SNP κ-CSN3 sobre o pico de produção, a produção inicial e a persistência das características qualitativas do leite. Já o SNP β-LG apresentou efeito significativo sobre os picos de produção de leite e gordura, e sobre o tempo necessário para atingir o pico de produção de proteína. A identificação e a estimação da influência dos marcadores SNP avaliados sobre as curvas de lactação e a qualidade do leite podem contribuir para a seleção de caprinos leiteiros.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

Frequencies of candidate genes and associations with carcass and meat traits in Nellore and crossbred cattle

Abstract: The objective of this work was to estimate allelic frequencies of the polymorphisms IGF2/MboII (G > T) of the insulin-like growth factor 2 (IGF2) gene, DQ499531.1:g.134A > T of the pro-melanin-concentrating hormone (PMCH) gene, and DQ667048.1:g.3290G > T of the RARrelated orphan receptor C (RORC) gene in beef cattle of different genetic groups, and to evaluate the associations between these polymorphisms and traits related to carcass composition and meat quality. Data on carcass and meat quality of 499 animals was used: of 313 Nellore (Bos indicus) and of 186 Nellore crossed with different taurine (Bos taurus) breeds. For the IGF2/MboII polymorphism, the frequencies found for the G allele were 0.231 and 0.631 for Nellore and crossed breeds, respectively. For the DQ499531.1:g.134A > T polymorphism, the allelic frequencies of A were 0.850 for Nellore and 0.905 for crossed breeds. For the DQ667048.1:g.3290G > T polymorphism, the allelic frequencies of G were 0.797 and 0.460 for Nellore and crossed breeds, respectively. The evaluated single nucleotide polymorphisms (SNPs) are not significantly associated with carcass and meat traits (rib eye area, back fat thickness, shear force, total lipids, and myofibrillar fragmentation index), suggesting little utility of the analyzed polymorphisms of the IGF2, PMHC, and RORC genes as selection markers in the studied cattle populations.

Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery.

The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Potential impact of environmental bacteriophages in spreading antibiotic resistance genes

The idea that bacteriophage transduction plays a role in the horizontal transfer of antibiotic resistance genes is gaining momentum. Such transduction might be vital in horizontal transfer from environmental to human bodyassociated biomes and here we review many lines of evidence supporting this notion. It is well accepted that bacteriophages are the most abundant entities in most environments, where they have been shown to be quite persistent. This fact, together with the ability of many phages to infect bacteria belonging to different taxa, makes them suitable vehicles for gene transfer. Metagenomic studies confirm that substantial percentages of the bacteriophage particles present in most environments contain bacterial genes, including mobile genetic elements and antibiotic resistance genes. When specific genes of resistance to antibiotics are detected by real-time PCR in the bacteriophage populations of different environments, only tenfold lower numbers of these genes are observed, compared with those found in the corresponding bacterial populations. In addition, the antibiotic resistance genes from these bacteriophages are functional and generate resistance to the bacteria when these genes are transfected. Finally, reports about the transduction of antibiotic resistance genes are on the increase.

A microarray-based system for the simultaneous analysis of single nucleotide polymorphisms in human genes involved in the metabolism of anti-malarial drugs

Background: In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR) fragments to detect single nucleotide polymorphisms (SNPs) in a larger number of samples. Methods: The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP) and N-acetyltransferase-2 (NAT2) involved in antimalarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. Results: For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00), whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00) was found, e. g. CYP2D6*17 (2850C>T), CYP3A4*1B and CYP3A5*3. Conclusion: The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity.

Pseudoachondroplasia and multiple epiphyseal dysplasia: a 7-year comprehensive analysis of the known disease genes identify novel and recurrent mutations and provides an accurate assessment of their relative contribution.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED.

Short communication. SNP analyses of the 5HT1R and STAT genes in Pacific white shrimp, Litopenaeus vannamei

La industria de la producción de camarón es una de las industrias acuícolas que se encuentra en más crecimiento en la actualidad. Los estudios para encontrar marcadores genéticos son muy efectivos para la mejora de sus propiedades y de gran interés para los productores de camarón. En este trabajo se utilizaron seis individuos de una población de Litopenaeus vannamei, donde se encontraron cuatro polimorfismos de nucleótido único (SNPs) en el gen 5HT1R (5-hidroxitriptamina receptor1) y un SNP en el gen STAT (transductor de señal y activador de la transcripción). Sin embargo, el polimorfismo en el gen STAT resultó ser homocigoto en una población diferente utilizada para análisis de asociación. Los presentes análisis revelaron que el alelo C, en dos polimorfismos SNP (C109T y C395G) del gen 5HT1R, tiende a estar asociado con el aumento del peso corporal. Consideramos que hay necesidad de hacer nuevos estudios utilizando una muestra más amplia y diversa de la población en cuestión.