Investigation of the role of ENPP1 and TNAP genes in chondrocalcinosis

Objectives. Extracellular inorganic pyrophosphate (ePPi) inhibits certain forms of pathological mineralization while promoting others. Three molecules involved in ePPi regulation are important candidates for the development of calcium pyrophosphate dihydrate chondrocalcinosis (CPPD CC). These include ANKH, ectonucleotide pyrophosphatase (ENPP1) and TNAP. We have previously showed that genetic variation in ANKH is a cause of autosomal dominant familial CC and also some sporadic cases of CPPD CC. We now investigate the possible role of ENPP1 and TNAP in CPPD CC. Methods. Exons, untranslated regions (UTR) and exon-intron boundaries of ENPP1 and TNAP were sequenced using ABI Big Dye chemistry on automated sequencers. Sixteen variants were identified (3 in ENPP1 and 13 in TNAP) and were subsequently genotyped in 128 sporadic Caucasian CPPD CC patients and 600 healthy controls using a combination of polymerase chain reaction/restriction fragment-length polymorphism analysis or using Taqman. Allele and genotype frequencies were compared between cases and controls using the χ 2 test. Linkage disequilibrium, haplotype and the single nucleotide polymorphism-specific analyses were also performed. This study had 80% power to detect an odds ratio of 2.2 or more at these loci. Results. No difference was observed in the allele or genotype frequencies between patients and controls at either ENPP1 or TNAP. Conclusions. Polymorphisms of ENPP1 and TNAP are not major determinants of susceptibility to CC in the population studied. Further studies of the aetiology of sporadic CPPD CC are required to determine its causes.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

Investigação de variantes exônicas nos genes VPS35, EIF4G1 e LRRK2 como causa da doença de Parkinson em casuística brasileira

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente no mundo, afetando 1-2% da população acima de 65 anos, caracterizada clinicamente por tremor em repouso, bradicinesia, instabilidade postural e rigidez muscular. Essas manifestações surgem devido à degeneração neuronal progressiva e à presença de inclusões proteicas ricas em α-sinucleína. A DP é decorrente da interação entre fatores ambientais e genéticos, e entre os fatores genéticos, variantes exônicas de transmissão dominante nos genes LRRK2 (leucine-rich repeat kinase 2), VPS35 (vacuolar protein sorting 35) e EIF4G1 (eukaryotic translation initiation factor 4-gamma 1) têm sido associadas à etiologia da doença. Entretanto, estudos sobre o efeito dessas variantes na população brasileira são raros ou inexistentes. Por essa razão, neste trabalho rastreamos mutações nos genes VPS35 (p.D620N; p.R524W), EIF4G1 (p.R1205H; p.A502V) e LRRK2 (p.G2019S) em uma amostra de 582 pacientes brasileiros com DP não aparentados e 329 indivíduos controles saudáveis. Além disso, conduzimos o primeiro estudo caso-controle para análise de variantes exônicas raras (p.Q1111H, p.T1410M, p.M1646T, p.S1761R, p.Y2189C) e comuns (p.N551K, p.R1398H, p.K1423K) no gene LRRK2 em um subgrupo de 329 pacientes brasileiros com DP, não aparentados, naturais da região sudeste. Esse subgrupo foi analisado e comparado com 222 indivíduos controles saudáveis a fim de verificar associações dessas variantes e a DP. Em relação às mutações dos genes VPS35 e EIF4G1, não foram encontradas alterações nos pacientes com DP. A mutação p.G2019S no gene LRRK2 foi encontrada em 15 probandos (2,6%), dos quais 9 são do sexo feminino (64,3%). O tremor em repouso foi observado em 47,36% dos pacientes com a mutação p.G2019S como primeiro sintoma motor. As análises das variantes raras no gene LRRK2 não revelaram qualquer associação estatisticamente significante. Entre as variantes comuns, a p.K1423K mostrou evidência de associação de risco com a DP (p<0,05) na estratificação contendo o grupo de indivíduos com história familiar da doença e para as variantes p.N551K e p.R1398H não foram observadas associações. A análise do haplótipo p.N551K-p.R1398H-p.K1423K revelou associação de proteção na amostra sudeste e na estratificação Rio de Janeiro (p<0,05). Esse haplótipo não está em desequilíbrio de ligação na amostra de 222 indivíduos controles brasileiros analisados (r2≤45). Os resultados obtidos neste estudo representam contribuições valiosas ao entendimento da relação entre as variantes genéticas estudadas e o risco de desenvolvimento da doença de Parkinson, principalmente no que se refere aos endofenótipos associados.

Molecular characterizations of oxytetracycline resistant bacteria and their resistance genes from mariculture waters of China

Oxytetracycline-resistant bacteria were isolated from a mariculture farm in China, and accounted for 32.23% and 5.63% of the total culturable microbes of the sea cucumber and the sea urchin rearing waters respectively. Marine vibrios, especially strains related to Vibrio splendidus or V. tasmaniensis, were the most abundant resistant isolates. For oxytetracycline resistance, tet(A), tet(B) and tet(D) genes were detected in both sea cucumber and sea urchin rearing ponds. The dominant resistance type for V. tasmaniensis-like strains was the combination of both tet(A) and tet(B) genes, while the major resistance type for V. splendidus-like strains was a single tet(D) gene. Most of the sea cucumber tet-positive isolates harbored a chloramphenicol-resistance gene, either cat IV or cat II, while only a few sea urchin tet-positive isolates harbored a cat gene, actually cat IV. The coexistence of tet and cat genes in the strains isolated from the mariculture farm studied was helpful in explaining some of the multi-resistance mechanisms. (c) 2006 Elsevier Ltd. All rights reserved.

Mapping the gene for autosomal dominant restless legs syndrome in an Irish family

Restless Legs Syndrome (RLS) is a common neurological disorder affecting nearly 15% of the general population. Ironically, RLS can be described as the most common condition one has never heard of. It is usually characterised by uncomfortable, unpleasant sensations in the lower limbs inducing an uncontrollable desire to move the legs. RLS exhibits a circadian pattern with symptoms present predominantly in the evening or at night, thus leading to sleep disruption and daytime somnolence. RLS is generally classified into primary (idiopathic) and secondary (symptomatic) forms. Primary RLS includes sporadic and familial cases of which the age of onset is usually less than 45 years and progresses slowly with a female to male ratio of 2:1. Secondary forms often occur as a complication of another health condition, such as iron deficiency or thyroid dysfunction. The age of onset is usually over 45 years, with an equal male to female ratio and more rapid progression. Ekbom described the familial component of the disorder in 1945 and since then many studies have been published on the familial forms of the disorder. Molecular genetic studies have so far identified ten loci (5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p). No specific gene within these loci has been identified thus far. Association mapping has highlighted a further five areas of interest. RLS6 has been found to be associated with SNPs in the BTBD9 gene. Four other variants were found within intronic and intergenic regions of MEIS1, MAP2K5/LBXCOR1, PTPRD and NOS1. The pathophysiology of RLS is complex and remains to be fully elucidated. Conditions associated with secondary RLS, such as pregnancy or end-stage renal disease, are characterised by iron deficiency, which suggests that disturbed iron homeostasis plays a role. Dopaminergic dysfunction in subcortical systems also appears to play a central role. An ongoing study within the Department of Pathology (University College Cork) is investigating the genetic characteristics of RLS in Irish families. A three generation RLS pedigree RLS3002 consisting of 11 affected and 7 unaffected living family members was recruited. The family had been examined for four of the known loci (5q, 12q, 14p and 9p) (Abdulrahim 2008). The aim of this study was to continue examining this Irish RLS pedigree for possible linkage to the previously described loci and associated regions. Using informative microsatellite markers linkage was excluded to the loci on 5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p and also within the regions reported to be associated with RLS. This suggested the presence of a new unidentified locus. A genome-wide scan was performed using two microsatellite marker screening sets (Research Genetics Inc. Mapping set and the Applied Biosystems Linkage mapping set version 2.5). Linkage analysis was conducted under an autosomal dominant model with a penetrance of 95% and an allele frequency of 0.01. A maximum LOD score of 3.59 at θ=0.00 for marker D19S878 indicated significant linkage on chromosome 19p. Haplotype analysis defined a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to 2.5 Mb. There are approximately 100 genes annotated within the critical region. Sequencing of two candidate genes, KLF16 and GAMT, selected on the assumed pathophysiology of RLS, did not identify any sequence variant. This study provides evidence of a novel RLS locus in an Irish pedigree, thus supporting the picture of RLS as a genetically heterogeneous trait.

Ets-1 p27: A novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51

The transcription factor Ets-1 is implicated in various physiological processes and invasive pathologies. We identified a novel variant of ets-1, ets-1Delta(III-VI), resulting from the alternative splicing of exons III to VI. This variant encodes a 27 kDa isoform, named Ets-1 p27. Ets-1 p27 lacks the threonine-38 residue, the Pointed domain and the transactivation domain, all of which are required for the transactivation of Ets-1 target genes. Both inhibitory domains surrounding the DNA-binding domain are conserved, suggesting that Ets-1 p27, like the full-length Ets-1 p51 isoform, is autoinhibited for DNA binding. We showed that Ets-1 p27 binds DNA in the same way as Ets-1 p51 does and that it acts both at a transcriptional and a subcellular localization level, thereby constituting a dual-acting dominant negative of Ets-1 p51. Ets-1 p27 blocks Ets-1 p51-mediated transactivation of target genes and induces the translocation of Ets-1 p51 from the nucleus to the cytoplasm. Furthermore, Ets-1 p27 overexpression represses the tumor properties of MDA-MB-231 mammary carcinoma cells in correlation with the known implication of Ets-1 in various cellular mechanisms. Thus the dual-acting dominant-negative function of Ets-1 p27 gives to the Ets-1 p27/Ets-1 p51 ratio a determining effect on cell fate.

Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation

Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Genetic diversity among Escherichia coli O157 : H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:117 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or H. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be 117) were significantly different from other Stx-positive and -negative E. coli O157:117 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:117 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.

Adaptive evolution of four microcephaly genes and the evolution of brain size in anthropoid primates

The anatomical basis and adaptive function of the expansion in primate brain size have long been studied; however, we are only beginning to understand the genetic basis of these evolutionary changes. Genes linked to human primary microcephaly have received much attention as they have accelerated evolutionary rates along lineages leading to humans. However, these studies focus narrowly on apes, and the link between microcephaly gene evolution and brain evolution is disputed. We analyzed the molecular evolution of four genes associated with microcephaly (ASPM, CDK5RAP2, CENPJ, MCPH1) across 21 species representing all major clades of anthropoid primates. Contrary to prevailing assumptions, positive selection was not limited to or intensified along the lineage leading to humans. In fact we show that all four loci were subject to positive selection across the anthropoid primate phylogeny. We developed clearly defined hypotheses to explicitly test if selection on these loci was associated with the evolution of brain size. We found positive relationships between both CDK5RAP2 and ASPM and neonatal brain mass and somewhat weaker relationships between these genes and adult brain size. In contrast, there is no evidence linking CENPJ and MCPH1 to brain size evolution. The stronger association of ASPM and CDK5RAP2 evolution with neonatal brain size than with adult brain size is consistent with these loci having a direct effect on prenatal neuronal proliferation. These results suggest that primate brain size may have at least a partially conserved genetic basis. Our results contradict a previous study that linked adaptive evolution of ASPM to changes in relative cortex size; however, our analysis indicates that this conclusion is not robust. Our finding that the coding regions of two widely expressed loci has experienced pervasive positive selection in relation to a complex, quantitative developmental phenotype provides a notable counterexample to the commonly asserted hypothesis that cisregulatory regions play a dominant role in phenotypic evolution. Key words: ASPM, MCPH1, CDK5RAP2, CENPJ, brain, neurogenesis, primates.

Modelling negative feedback networks for activating transcription factor 3 predicts a dominant role for miRNAs in immediate early gene regulation

Activating transcription factor 3 (Atf3) is rapidly and transiently upregulated in numerous systems, and is associated with various disease states. Atf3 is required for negative feedback regulation of other genes, but is itself subject to negative feedback regulation possibly by autorepression. In cardiomyocytes, Atf3 and Egr1 mRNAs are upregulated via ERK1/2 signalling and Atf3 suppresses Egr1 expression. We previously developed a mathematical model for the Atf3-Egr1 system. Here, we adjusted and extended the model to explore mechanisms of Atf3 feedback regulation. Introduction of an autorepressive loop for Atf3 tuned down its expression and inhibition of Egr1 was lost, demonstrating that negative feedback regulation of Atf3 by Atf3 itself is implausible in this context. Experimentally, signals downstream from ERK1/2 suppress Atf3 expression. Mathematical modelling indicated that this cannot occur by phosphorylation of pre-existing inhibitory transcriptional regulators because the time delay is too short. De novo synthesis of an inhibitory transcription factor (ITF) with a high affinity for the Atf3 promoter could suppress Atf3 expression, but (as with the Atf3 autorepression loop) inhibition of Egr1 was lost. Developing the model to include newly-synthesised miRNAs very efficiently terminated Atf3 protein expression and, with a 4-fold increase in the rate of degradation of mRNA from the mRNA/miRNA complex, profiles for Atf3 mRNA, Atf3 protein and Egr1 mRNA approximated to the experimental data. Combining the ITF model with that of the miRNA did not improve the profiles suggesting that miRNAs are likely to play a dominant role in switching off Atf3 expression post-induction.

Identification and expression analyses of Cytosolic Glutamine Synthetase genes in barley (Hordeum vulgare L.)

Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, particularly during seed development. Three cytosolic GS isoforms (HvGS1) were identified in barley (Hordeum vulgare L. cv Golden Promise). Quantitation of gene expression, localization and response to N supply revealed that each gene plays a non-redundant role in different tissues and during development. Localization of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that it is important in N transport and remobilization. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localized to the leaf mesophyll cells, in the cortex and pericycle of roots, and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in leaves with an increasing supply of N, suggesting its role in the primary assimilation of N. HvGS1_3 was specifically and predominantly localized in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH+4, suggesting that it has a primary role in grain N assimilation and also in the protection against ammonium toxicity in roots. The expression of HvGS1 genes is directly correlated with protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley.

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

A new locus for autosomal dominant amelogenesis imperfecta on chromosome 8q24.3

Amelogenesis imperfecta (AI) is a collective term used to describe phenotypically diverse forms of defective tooth enamel development. AI has been reported to exhibit a variety of inheritance patterns, and several loci have been identified that are associated with AI. We have performed a genome-wide scan in a large Brazilian family segregating an autosomal dominant form of AI and mapped a novel locus to 8q24.3. A maximum multipoint LOD score of 7.5 was obtained at marker D8S2334 (146,101,309 bp). The disease locus lies in a 1.9 cM (2.1 Mb) region according to the Rutgers Combined Linkage-Physical map, between a VNTR marker (at 143,988,705 bp) and the telomere (146,274,826 bp). Ten candidate genes were identified based on gene ontology and microarray-facilitated gene selection using the expression of murine orthologues in dental tissue, and examined for the presence of a mutation. However, no causative mutation was identified.

Associação de polimorfismos nos genes TLR2 e TLR4 com risco de câncer colorretal esporádico e influência na expressão gênica

Pós-graduação em Genética - IBILCE