Identification of IL6R and chromosome 11q13.5 as risk loci for asthma

We aimed to identify novel genetic variants affecting asthma risk, since these might provide novel insights into molecular mechanisms underlying the disease. We did a genome-wide association study (GWAS) in 2669 physician-diagnosed asthmatics and 4528 controls from Australia. Seven loci were prioritised for replication after combining our results with those from the GABRIEL consortium (n=26 475), and these were tested in an additional 25 358 independent samples from four in-silico cohorts. Quantitative multi-marker scores of genetic load were constructed on the basis of results from the GABRIEL study and tested for association with asthma in our Australian GWAS dataset. Two loci were confirmed to associate with asthma risk in the replication cohorts and reached genome-wide significance in the combined analysis of all available studies (n=57 800): rs4129267 (OR 1·09, combined p= 2·4×10-8) in the interleukin-6 receptor (IL6R) gene and rs7130588 (OR 1·09, p=1·8×10-8) on chromosome 11q13.5 near the leucine-rich repeat containing 32 gene (LRRC32, also known as GARP). The 11q13.5 locus was significantly associated with atopic status among asthmatics (OR 1·33, p=7×10-4), suggesting that it is a risk factor for allergic but not non-allergic asthma. Multi-marker association results are consistent with a highly polygenic contribution to asthma risk, including loci with weak effects that might be shared with other immune-related diseases, such as NDFIP1, HLA-B, LPP, and BACH2. The IL6R association further supports the hypothesis that cytokine signalling dysregulation affects asthma risk, and raises the possibility that an IL6R antagonist (tocilizumab) may be effective to treat the disease, perhaps in a genotype-dependent manner. Results for the 11q13.5 locus suggest that it directly increases the risk of allergic sensitisation which, in turn, increases the risk of subsequent development of asthma. Larger or more functionally focused studies are needed to characterise the many loci with modest effects that remain to be identified for asthma. National Health and Medical Research Council of Australia. A full list of funding sources is provided in the webappendix. © 2011 Elsevier Ltd.

Loci affecting gamma-glutamyl transferase in adults and adolescents show age x SNP interaction and cardiometabolic disease associations

Serum gamma-glutamyl transferase (GGT) activity is a marker of liver disease which is also prospectively associated with the risk of all-cause mortality, cardiovascular disease, type 2 diabetes and cancers. We have discovered novel loci affecting GGT in a genome-wide association study (rs1497406 in an intergenic region of chromosome 1, P = 3.9 x 10(-8); rs944002 in C14orf73 on chromosome 14, P = 4.7 x 10(-13); rs340005 in RORA on chromosome 15, P = 2.4 x 10(-8)), and a highly significant heterogeneity between adult and adolescent results at the GGT1 locus on chromosome 22 (maximum P(HET) = 5.6 x 10(-12) at rs6519520). Pathway analysis of significant and suggestive single-nucleotide polymorphism associations showed significant overlap between genes affecting GGT and those affecting common metabolic and inflammatory diseases, and identified the hepatic nuclear factor (HNF) family as controllers of a network of genes affecting GGT. Our results reinforce the disease associations of GGT and demonstrate that control by the GGT1 locus varies with age.

Association between ORMDL3, IL1RL1 and a deletion on chromosome 17q21 with asthma risk in Australia

Genome-wide association studies followed by replication provide a powerful approach to map genetic risk factors for asthma. We sought to search for new variants associated with asthma and attempt to replicate the association with four loci reported previously (ORMDL3, PDE4D, DENND1B and IL1RL1). Genome-wide association analyses of individual single nucleotide polymorphisms (SNPs), rare copy number variants (CNVs) and overall CNV burden were carried out in 986 asthma cases and 1846 asthma-free controls from Australia. The most-associated locus in the SNP analysis was ORMDL3 (rs6503525, P = 4.8 x 10(-)(7)). Five other loci were associated with P < 10(-)(5), most notably the chemokine CXC motif ligand 14 (CXCL14) gene (rs31263, P = 7.8 x 10(-)(6)). We found no evidence for association with the specific risk variants reported recently for PDE4D, DENND1B and ILR1L1. However, a variant in IL1RL1 that is in low linkage disequilibrium with that reported previously was associated with asthma risk after accounting for all variants tested (rs10197862, gene wide P = 0.01). This association replicated convincingly in an independent cohort (P = 2.4 x 10(-)(4)). A 300-kb deletion on chromosome 17q21 was associated with asthma risk, but this did not reach experiment-wide significance. Asthma cases and controls had comparable CNV rates, length and number of genes affected by deletions or duplications. In conclusion, we confirm the association between asthma risk and variants in ORMDL3 and identify a novel risk variant in IL1RL1. Follow-up of the 17q21 deletion in larger cohorts is warranted.

Susceptibility variants for male-pattern baldness on chromosome 20p11

We carried out a genome-wide association study in 296 individuals with male-pattern baldness (androgenetic alopecia) and 347 controls. We then investigated the 30 best SNPs in an independent replication sample and found highly significant association for five SNPs on chromosome 20p11 (rs2180439 combined P = 2.7 x 10(-15)). No interaction was detected with the X-chromosomal androgen receptor locus, suggesting that the 20p11 locus has a role in a yet-to-be-identified androgen-independent pathway.

A model for mammalian male determination based on a passive Y chromosome

A model is suggested for mammalian male determination based on interactions postulated to occur among an autosomal repressor gene, an X-linked male-determining gene termed Tdx, and multiple copies of certain DNA sequences on the Y chromosome that do not code for any protein. The repressor, synthesised in limited amounts, has higher affinity for the Y-linked sequences than for Tdx and its affinity for Tdx is greater than that of RNA polymerase. In XY cells the Y effectively binds all available repressor, permitting transcription of Tdx to occur. In XX cells, since competition from the Y-linked high-affinity sequences is absent, the repressor binds to Tdx and prevents transcription. As a result of this competition between Tdx and the Y-linked high-affinity sites for limiting concentrations of the autosomal repressor, the product of the Tdx gene (TDX) is synthesized in the male but not in the female. It is suggested that in determination of the male sex, the role of the Y chromosome is to serve as a sink for the Tdx repressor. The proposed interactions provide a plausible explanation for the genetic properties of several anomalies of sexual development in mouse, man, and other mammals. The model suggests that the postulated multiple, highaffinity sequences on the Y chromosome of the mouse are included among the DNA sequences referred to as the Sxr-Bkm sequences.

The evolution of genomic imprinting

We explore three possible pathways for the evolution of genomic imprinting. (1) Imprinting may be advantageous in itself when imprinted and unimprinted alleles of a locus confer different phenotypes. If a segment of DNA is imprinted in the gametes of one sex but not in those of the other, it might lead to effects correlated with sexual dimorphism. More fundamentally, in certain organisms, sex determination might have evolved because of imprinting. When imprinting leads to chromosome elimination or inactivation and occurs in some embryos but not in others, two classes of embryos, differing in the number of functional gene copies, would result. A model for sex determination based on inequality in the actual or effective copy-number of particular noncoding, regulatory sequences of DNA has been proposed (Chandra, Proc. natn. Acad. Sci. U.S.A. 82. 1165–1169 and 6947–6949, 1985). Maternal control of offspring sex is another possible consequence of imprinting; this would indicate a potential role for imprinting in sex ratio evolution. (2) Genes responsible for imprinting may have pleiotropic effects and they may have been selected for reasons other than their imprinting ability. Lack of evidence precludes further consideration of this possibility. (3) Imprinting could have co-evolved with other traits. For instance, gamete-specific imprinting could lead to a lowered fitness of androgenetic or gynogenetic diploids relative to the fitness of ‘normal’ diploids. This in turn would reinforce the evolution of anisogamy. The reversibility of imprinting raises the possibility of occasional incomplete or improper erasure. If the site of imprinting is the egg – as appears to be the case with the human X (Chandra and Brown, Nature 253. 165–168, 1975) – either improper imprinting or improper erasure could lead to unusual patterns of inheritance (as in the fragile-X syndrome) or fitness effects skipping generations.

Novel Rearrangements in the Staphylococcal Cassette Chromosome Mec Type V Elements of Indian ST772 and ST672 Methicillin Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a commensal gram positive bacteria which causes severe and non severe infections in humans and livestock. In India, ST772 is a dominant and ST672 is an emerging clone of Staphylococcus aureus. Both cause serious human diseases, and carry type V SCCmec elements. The objective of this study was to characterize SCCmec type V elements of ST772 and ST672 because the usual PCR methods did not amplify all primers specific to the type. Whole genome sequencing analysis of seven ST772 and one ST672 S. aureus isolates revealed that the SCCmec elements of six of the ST772 isolates were the smallest of the extant type V elements and in addition have several other novel features. Only one ST772 isolate and the ST672 isolate carried bigger SCCmec cassettes which were composites carrying multiple ccrC genes. These cassettes had some similarities to type V SCCmec element from M013 isolate (ST59) from Taiwan in certain aspects. SCCmec elements of all Indian isolates had an inversion of the mec complex, similar to the bovine SCCmec type X. This study reveals that six out of seven ST772 S. aureus isolates have a novel type V (5C2) SCCmec element while one each of ST772 and ST672 isolates have a composite SCCmec type V element (5C2&5) formed by the integration of type V SCCmec into a MSSA carrying a SCC element, in addition to the mec gene complex inversions and extensive recombinations.

Photocatalytic inactivation of E-Coli by ZnO-Ag nanoparticles under solar radiation

Porous and fluffy ZnO photocatalysts were successfully prepared via simple solution based combustion synthesis method. The photocatalytic inactivation of Escherichia coli bacteria was studied separately for both Ag substituted and impregnated ZnO under irradiation of natural solar light. A better understanding of substitution and impregnation of Ag was obtained by Raman spectrum and X-ray photoelectron analysis. The reaction parameters such as catalyst dose, initial bacterial concentration and effect of hydroxyl radicals via H2O2 addition were also studied for ZnO catalyst. Effective inactivation was observed with 0.25 g L-1 catalyst loading having 10(9) CFU mL(-1) bacterial concentration. With an increase in molarity of H2O2, photocatalytic inactivation was enhanced. The effects of different catalysts were studied, and highest bacterial killing was observed by Ag impregnated ZnO with 1 atom% Ag compared to Ag substituted ZnO. This enhanced activity can be attributed to effective charge separation that is supported by photoluminescence studies. The kinetics of reaction in the presence of different scavengers showed that reaction is significantly influenced by the presence of hole and hydroxyl radical scavenger with high efficiency.

Desenvolvimento e validação de sistema multiplex X-STR para identificação humana por DNA

Novas metodologias de análise molecular voltadas para estudos populacionais, clínicos, evolutivos, da biodiversidade e identificação forense foram desenvolvidas com base em marcadores microssátelites ou STR Short Tandem Repeats. Os marcadores STR, que estão amplamente espalhados nos genomas e se caracterizam por apresentar alto grau de polimorfismo, podem ser analisados a partir da amplificação por PCR (Reação em Cadeia da polimerase). A análise foi facilitada a partir do desenvolvimento de sistemas de amplificação simultânea de múltiplos STR (multiplex STR) e com a detecção automatizada dos produtos de amplificação marcados por fluorescência. Recentemente, o uso de marcadores STR do cromossomo X (X-STR) tornou-se significativo na prática forense. Devido ao seu modo de transmissão, os X-STR são úteis em situações particulares de investigação de relações de parentesco, apresentando vantagens sobre o uso de STR autossômicos. Este estudo teve como principal objetivo o desenvolvimento e validação de sistema multiplex, denominado LDD (X-STR) Decaplex, capaz de amplificar dez loci X-STR (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 e DXS10101) para aplicação em genética populacional, identificação e análises forenses. Utilizando o LDD (X-STR) Decaplex 170 indivíduos autodenominados afrodescendentes, não aparentados geneticamente, foram genotipados. As freqüências alélicas e genotípicas não apresentaram desvio do equilíbrio de Hardy-Weinberg e estão em concordância com aquelas observadas em outros estudos. Os haplótipos observados foram únicos em indivíduos de amostra masculina. A análise de desequilíbrio de ligação não revelou associação entre os marcadores X-STR. A diversidade genética foi elevada, variando entre 0,6218 para o locus DXS7133 a 0,9327 para o locus DXS8377. Os parâmetros de Probabilidade de Vinculação (PV), Índice de Vinculação (IV), Poder de Exclusão (PE), Poder de Discriminação e Razão de Verossimilhança foram também elevados, demonstraram que os dez X-STRs são altamente polimórficos e discriminativos na população estudada. A concentração mínima de DNA para a amplificação dos loci do LDD (X-STR) Decaplex é de 0,5 ng e verificamos que amplificação por PCR pode ser afetada quando são adicionados mais de 5 ng de DNA nas reações. Os percentuais de bandas stutter foram elevados para os loci DXS7132 e DXS8377. No teste de reprodutibilidade observamos consistência entre as tipagem de diferentes amostras biológicas, incluindo as de restos mortais. No teste de mistura a proporção limite em que observamos a coexistência de duas espécies biológicas foi de 2,5:1ng (feminino-masculino). Os resultados evidenciaram que os loci do LDD (X-STR) Decaplex são altamente informativos, consistindo, em conjunto, uma ferramenta importante em estudos de identificação humana e de relações de parentesco.

Investigação de alterações em genes de microRNAs expressos no cérebro como causa de deficiência intelectual ligada ao cromossomo X

A Deficiência Intelectual (DI) é uma condição definida como um funcionamento intelectual significativamente prejudicado, expresso juntamente com limitações em pelo menos duas áreas do comportamento adaptativo que se manifestam antes dos 18 anos de idade. A prevalência estimada da DI na população em geral é de 2-3% e um número expressivo de casos permanece sem um diagnóstico definitivo. Há um consenso geral de que a DI é mais comum em indivíduos do sexo masculino em relação aos do sexo feminino. Entre as explicações para este excesso está a concentração de genes específicos para a habilidade cognitiva no cromossomo X. MicroRNAs (miRNAs) são pequenas moléculas de RNA não codificador que modulam a expressão gênica pós-transcricional de RNAs mensageiros alvo. Recentemente, estudos têm demonstrado a importância essencial dos miRNAs para o desenvolvimento e funcionamento cerebrais e sabe-se que o cromossomo X tem uma alta densidade de genes de miRNAs. Neste contexto, os miRNAs são candidatos potenciais como fatores genéticos envolvidos na Deficiência Intelectual Ligada ao X (DILX). Neste estudo, foram analisadas as regiões genômicas de 17 genes de miRNAs expressos no cérebro localizados no cromossomo X, com o objetivo de investigar o possível envolvimento de variantes na sequência destes miRNAs na DILX. Para este fim, selecionamos amostras de DNA genômico (sangue periférico) de 135 indivíduos do sexo masculino portadores de DI sugestiva de DILX de um grupo de mais de 1.100 pacientes com DI encaminhados ao Serviço de Genética Humana da UERJ. O critério de inclusão para este estudo era de que os probandos apresentassem um ou mais parentes do sexo masculino afetados pela DI que fossem interligados por via materna. As amostras de DNA dos pacientes foram amplificadas utilizando a técnica de reação em cadeia da polimerase, seguida por purificação e sequenciamento direto pelo método de Sanger dos fragmentos amplificados. Para avaliar a conservação dos 17 miRNAs foi realizada uma análise filogenética in silico incluindo sequências dos miRNAs selecionados de humanos e de outras 8 espécies de primatas estreitamente relacionadas. Não foram encontradas alterações nas sequências nos genes de 17 miRNAs analisados, mesmo diante do padrão genético altamente heterogêneo da população brasileira. Adicionalmente, a análise filogenética destes miRNAs revelou uma alta conservação entre as espécies comparadas. Considerando o papel dos miRNAs como reguladores da expressão gênica, a ausência de alterações e a alta conservação entre primatas sugerem uma forte pressão seletiva sobre estas moléculas, reforçando a sua importância funcional para o organismo em geral. Apesar de não termos encontrado variantes de sequência nos miRNAs estudados, o envolvimento de miRNAs na DI não pode ser completamente descartado. Alterações fora da molécula de miRNA precursor, nos fatores de processamento, nos sítios alvo e variações no número de cópias de genes de miRNAs podem implicar em alteração na expressão dos miRNAs e, consequentemente, na funcionalidade do miRNA maduro. Sendo assim, uma análise sistemática da expressão de miRNAs em pacientes com DILX é urgentemente necessária, a fim de desvendar novos genes/mecanismos moleculares relacionados a esta condição.

Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human

Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats.

Chromosome painting in the long-tailed field mouse provides insights into the ancestral murid karyotype

We report on the hybridization of mouse chromosomal paints to Apodemus sylvaticus, the long-tailed field mouse. The mouse paints detected 38 conserved segments in the Apodemus karyotype. Together with the species reported here there are now six species of rodents mapped with Mus musculus painting probes. A parsimony analysis indicated that the syntenies of nine M. musculus chromosomes were most likely already formed in the muroid ancestor: 3, 4, 7, 9, 14, 18, 19, X and Y. The widespread occurrence of syntenic segment associations of mouse chromosomes 1/17, 2/13, 7/19, 10/17, 11/16, 12/17 and 13/15 suggests that these associations were ancestral syntenies for muroid rodents. The muroid ancestral karyotype probably had a diploid number of about 2n = 54. It would be desirable to have a richer phylogenetic array of species before any final conclusions are drawn about the Muridae ancestral karyotype. The ancestral karyotype presented here should be considered as a working hypothesis. Copyright (C) 2004 S. Karger AG, Basel.

Cross-species chromosome painting in the golden mole and elephant-shrew: support for the mammalian clades Afrotheria and Afroinsectiphillia but not Afroinsectivora

Cross-species painting (fluorescence in situ hybridization) with 23 human (Homo sapiens (HSA)) chromosome-specific painting probes (HSA 1-22 and the X) was used to delimit regions of homology on the chromosomes of the golden mole (Ghrysochloris asiaticus) and elephant-shrew (Elephantulus rupestris). A cladistic interpretation of our data provides evidence of two unique associations, HSA 1/19p and 5/21/3, that support Afrotheria. The recognition of HSA 5/3/21 expands on the 3/21 synteny originally designated as an ancestral state for all eutherians. We have identified one adjacent segment combination (HSA2/8p/4) that is supportive of Afroinsectiphillia (aardvark, golden mole, elephant-shrew). Two segmental combinations (HSA 10q/17 and HSA 3/20) unite the aardvark and elephant-shrews as sister taxa. The finding that segmental syntenies in evolutionarily distant taxa can improve phylogenetic resolution suggests that they may be useful for testing sequence-based phylogenies of the early eutherian mammals. They may even suggest clades that sequence trees are not recovering with any consistency and thus encourage the search for additional rare genomic changes among afrotheres.

Phylogenomic study of the subfamily Caprinae by cross-species chromosome painting with Chinese muntjac paints

Chromosomal homologies have been established between the Chinese muntjac (Muntiacus reevesi, MRE, 2n = 46) and five ovine species: wild goat (Capra aegagrus, CAE, 2n = 60), argall (Ovis ammon, OAM, 2n = 56), snow sheep (Ovis nivicola, ONI, 2n = 52), red goral (Naemorhedus cranbrooki, NCR, 2n = 56) and Sumatra serow (Capricornis sumatraensis, CSU, 2n = 48) by chromosome painting with a set of chromosome-specific probes of the Chinese muntjac. In total, twenty-two Chinese muntjac autosomal painting probes detected thirty-five homologous segments in the genome of each species. The chromosome X probe hybridized to the whole X chromosomes of all ovine species while the chromosome Y probe gave no signal. Our results demonstrate that almost all homologous segments defined by comparative painting show a high degree of conservation in G-banding patterns and that each speciation event is accompanied by specific chromosomal rearrangements. The combined analysis of our results and previous cytogenetic and molecular systematic results enables us to map the chromosomal rearrangements onto a phylogenetic tree, thus providing new insights into the karyotypic evolution of these species.

Karyotype relationships of six bat species (Chiroptera, Vespertilionidae) from China revealed by chromosome painting and G-banding comparison

The Vespertilionidae is the largest family in the order Chiroptera and has a worldwide distribution in the temperate and tropical regions. In order to further clarify the karyotype relationships at the lower taxonomic level in Vespertilionidae, genome-wid