The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.     

Uncovering hidden variance: pair-wise SNP analysis accounts for additional variance in nicotine dependence

Results from genome-wide association studies of complex traits account for only a modest proportion of the trait variance predicted to be due to genetics. We hypothesize that joint analysis of polymorphisms may account for more variance. We evaluated this hypothesis on a case–control smoking phenotype by examining pairs of nicotinic receptor single-nucleotide polymorphisms (SNPs) using the Restricted Partition Method (RPM) on data from the Collaborative Genetic Study of Nicotine Dependence (COGEND). We found evidence of joint effects that increase explained variance. Four signals identified in COGEND were testable in independent American Cancer Society (ACS) data, and three of the four signals replicated. Our results highlight two important lessons: joint effects that increase the explained variance are not limited to loci displaying substantial main effects, and joint effects need not display a significant interaction term in a logistic regression model. These results suggest that the joint analyses of variants may indeed account for part of the genetic variance left unexplained by single SNP analyses. Methodologies that limit analyses of joint effects to variants that demonstrate association in single SNP analyses, or require a significant interaction term, will likely miss important joint effects.     

Imputation across genotyping arrays for genome-wide association studies: assessment of bias and a correction strategy

A great promise of publicly sharing genome-wide association data is the potential to create composite sets of controls. However, studies often use different genotyping arrays, and imputation to a common set of SNPs has shown substantial bias: a problem which has no broadly applicable solution. Based on the idea that using differing genotyped SNP sets as inputs creates differential imputation errors and thus bias in the composite set of controls, we examined the degree to which each of the following occurs: (1) imputation based on the union of genotyped SNPs (i.e., SNPs available on one or more arrays) results in bias, as evidenced by spurious associations (type 1 error) between imputed genotypes and arbitrarily assigned case/control status; (2) imputation based on the intersection of genotyped SNPs (i.e., SNPs available on all arrays) does not evidence such bias; and (3) imputation quality varies by the size of the intersection of genotyped SNP sets. Imputations were conducted in European Americans and African Americans with reference to HapMap phase II and III data. Imputation based on the union of genotyped SNPs across the Illumina 1M and 550v3 arrays showed spurious associations for 0.2 % of SNPs: ~2,000 false positives per million SNPs imputed. Biases remained problematic for very similar arrays (550v1 vs. 550v3) and were substantial for dissimilar arrays (Illumina 1M vs. Affymetrix 6.0). In all instances, imputing based on the intersection of genotyped SNPs (as few as 30 % of the total SNPs genotyped) eliminated such bias while still achieving good imputation quality.     

A meta-analysis of two genome-wide association studies to identify novel loci for maximum number of alcoholic drinks

Maximum number of alcoholic drinks consumed in a 24-h period (maxdrinks) is a heritable (>50 %) trait and is strongly correlated with vulnerability to excessive alcohol consumption and subsequent alcohol dependence (AD). Several genome-wide association studies (GWAS) have studied alcohol dependence, but few have concentrated on excessive alcohol consumption. We performed two GWAS using maxdrinks as an excessive alcohol consumption phenotype: one in 118 extended families (N = 2,322) selected from the Collaborative Study on the Genetics of Alcoholism (COGA), and the other in a case–control sample (N = 2,593) derived from the Study of Addiction: Genes and Environment (SAGE). The strongest association in the COGA families was detected with rs9523562 (p = 2.1 × 10^?6) located in an intergenic region on chromosome 13q31.1; the strongest association in the SAGE dataset was with rs67666182 (p = 7.1 × 10^?7), located in an intergenic region on chromosome 8. We also performed a meta-analysis with these two GWAS and demonstrated evidence of association in both datasets for the LMO1 (p = 7.2 × 10^?7) and PLCL1 genes (p = 4.1 × 10^?6) with maxdrinks. A variant in AUTS2 and variants in INADL, C15orf32 and HIP1 that were associated with measures of alcohol consumption in a meta-analysis of GWAS studies and a GWAS of alcohol consumption factor score also showed nominal association in the current meta-analysis. The present study has identified several loci that warrant further examination in independent samples. Among the top SNPs in each of the dataset (p ≤ 10^?4) far more showed the same direction of effect in the other dataset than would be expected by chance (p = 2 × 10^?3, 3 × 10^?6), suggesting that there are true signals among these top SNPs, even though no SNP reached genome-wide levels of significance.     

Differential expression of PARK2 splice isoforms in an in vitro model of dopaminergic‐like neurons exposed to toxic insults mimicking Parkinson's disease

Mutations in PARK2 (or parkin) are responsible for 50% of cases of autosomal‐recessive juvenile‐onset Parkinson's disease (PD). To date, 21 alternative splice variants of the human gene have been cloned. Yet most studies have focused on the full‐length protein, whereas the spectrum of the parkin isoforms expressed in PD has never been investigated. In this study, the role of parkin proteins in PD neurodegeneration was explored for the first time by analyzing their expression profile in an in vitro model of PD. To do so, undifferentiated and all‐trans‐retinoic‐acid (RA)‐differentiated SH‐SY5Y cells (which thereby acquire a PD‐like phenotype) were exposed to PD‐mimicking neurotoxins: 1‐methyl‐4‐phenylpyridinium (MPP⁺) and 6‐hydroxydopamine (6‐OHDA) are widely used in PD models, whereas carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) and carbobenzoxy‐Leu‐Leu‐leucinal (MG132) interfere, respectively, with mitochondrial mitophagy and proteasomal degradation. Following treatment with each neurotoxin H1, the first parkin isoform to be cloned, was down‐regulated compared to the respective controls both in undifferentiated and RA‐differentiated cells. In contrast, the expression pattern of the minor splice isoforms varied as a function of the compound used: it was largely unchanged in both cell cultures (eg, H21‐H6, H12, XP isoform) or it showed virtually opposite alterations in undifferentiated and RA‐differentiated cells (eg, H20 and H3 isoform). This complex picture suggests that up‐ or down‐regulation may be a direct effect of toxin exposure, and that the different isoforms may exert different actions in neurodegeneration via modulation of different molecular pathways.     

MmtDB: a Metazoa mitochondrial DNA variants database.

The present paper describes the structure of MmtDB-a specialized database designed to collect Metazoa mitochondrial DNA variants. Priority in the data collection is given to the Metazoa species for which a large amount of variants is available, as it is the case for human variants. Starting from the sequences available in the Nucleotide Sequence Databases, the redundant sequences are removed and new sequences from other sources are added. Value-added information are associated to each variant sequence, e.g. analysed region, experimental method, tissue and cell lines, population data, sex, age, family code and information about the variation events (nucleotide position, involved gene, restriction site's gain or loss). Cross-references are introduced to the EMBL Data Library, as well as an internal cross-referencing among MmtDB entries according to their tissual, heteroplasmic, familiar and aplotypical correlation. MmtDB can be accessed through the World Wide Web at URL [see text].     

Molecular evolution of mammalian aquaporin-2: further evidence that elephant shrew and aardvark join the paenungulate clade

A 328-bp sequence from exon 1 of the gene for aquaporin-2 (AQP2) was compared in 12 mammalian species, representing as many eutherian orders. This sequence encodes the N-terminal half of this kidney- specific water channel protein. Most amino acid replacements, as well as an insertion, have occurred in extracellular loops connecting the transmembrane helices, in agreement with a lower functional importance of these loops. Phylogenetic analyses were performed with parsimony, distance, and maximum-likelihood methods. The AQP2 data set, alone as well as in combination with previously published alpha A-crystallin protein sequences, strongly supports a clade consisting of elephant, hyrax, aardvark, and elephant shrew, reaching bootstrap values of 99%. This finding fully agrees with the only other presently available sequence data sets that include these taxa, those of von Willebrand factor and interphotoreceptor retinoid-binding protein, and suggests that this extended paenungulate clade is one of the most conspicuous superordinal groupings in eutherian phylogeny. Some support was obtained for an artiodactyl/perissodactyl clade, while the grouping of pholidotes with edentates was contradicted.      

Use of Femoral Head and Neck Ostectomy and Physical Therapy to Manage Osteoarthritis in a Rhesus Macaque (Macaca mulatta)

Osteoarthritis is associated with pain and immobility in both humans and animals. However, available resources for osteoarthritis management in captive NHP are limited. This case report describes a novel management strategy for a 10-y-old male macaque with unilateral hindlimb lameness, prominent muscle wasting, and severely limited range of motion. Radiographs of the affected limb showed lytic lesions of the femoral head. To relieve pain and improve mobility, femoral head and neck ostectomy (FHO) was performed, and multiple pharmacotherapies were initiated. The macaque also received a unique method of physical therapy that required no sedation, acted as enrichment, and was implemented by using a conventional caging system. The response to therapy was monitored by measuring thigh circumference in the operated and nonoperated limbs, which demonstrated improvement in both legs. The unique physical therapy in conjunction with surgery and pharmacotherapy benefited the macaque with osteoarthritis by reducing discomfort and improving mobility.Abbreviations: FHO, femoral head and neck ostectomy; ROM, range of motionOsteoarthritis is associated with pain and is the leading cause of impaired mobility in aging humans.⁷ Osteoarthritis also is frequently diagnosed in companion animals, and the most common presenting complaint is lameness.⁴ Physical examination findings may include lameness, reduced range of motion (ROM), palpable crepitus, joint instability, and pain during joint manipulation.⁴ In captive NHP, clinical signs may be limited to muscle atrophy,¹² in part because of their natural instincts, as wild animals and as prey species, to hide injury and pain.¹In companion animals, pain from osteoarthritis frequently is managed with a combination of drugs including NSAID, nutritional supplements, and weight reduction.¹¹ If an underlying cause is identified, surgery may be indicated. In addition, physical rehabilitation including ROM exercises, underwater treadmill walking, low-level laser therapy, and acupuncture may be beneficial in managing pain due to osteoarthritis.¹¹Various challenges are associated with managing osteoarthritis in captive NHP. Due to biosafety concerns, physical rehabilitation exercises developed for companion animals are often not feasible. Physical manipulation generally requires chemical sedation; sedation itself may adversely affect hemodynamic and respiratory functions, with an increase in frequency of sedation leading to increased risks.¹³ The chronic use of antiinflammatory medications such as NSAID may be associated with gastrointestinal, hepatic, and renal adverse effects. In addition, published information describing medical and surgical approaches for NHP, especially in regard to postoperative care, is limited.^3,5,12,15This case report involves an adult male rhesus macaque with osteoarthritis that led to unilateral hindlimb lameness and prominent muscle wasting. The macaque was managed with a combination of pharmacotherapy, surgery (femoral head and neck ostectomy [FHO]), and innovative physical therapy.