Testing whether macroevolution follows microevolution: Are colour differences among swans (<Emphasis Type="Italic">Cygnus</Emphasis>) attributable to variation at the <Emphasis Type="Italic">MC1R</Emphasis> locus?

Background  

The MC1R (melanocortin-1 receptor) locus underlies intraspecific variation in melanin-based dark plumage coloration in several unrelated birds with plumage polymorphisms. There is far less evidence for functional variants of MC1R being involved in interspecific variation, in which spurious genotype-phenotype associations arising through population history are a far greater problem than in intraspecific studies. We investigated the relationship between MC1R variation and plumage coloration in swans (Cygnus), which show extreme variation in melanic plumage phenotypes among species (white to black).     

Mutation analysis of the <Emphasis Type="Italic">COL1A1</Emphasis> and <Emphasis Type="Italic">COL1A2</Emphasis> genes in Vietnamese patients with osteogenesis imperfecta

Background

The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI.

Methods

Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish’s osteogenesis imperfecta mutation database.

Results

The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G?>?A (p.Gly821Ser) in four unrelated patients and one, c.2005G?>?A (p.Ala669Thr), in two unrelated patients.

Conclusion

Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.

     

Genotype frequencies and linkage disequilibrium in the CEPH human diversity panel for variants in folate pathway genes MTHFR, MTHFD, MTRR, RFC1, and GCP2

BACKGROUND

Genetic variation in enzymes involved in vitamin metabolism is a candidate for analysis in studies of how nutritional covariates may impact a disease state. The role of folate pathway genes in birth defects and cardiovascular disease in humans has been widely studied. Since incidence rates for these disorders vary by geographic origins, it is useful to know which variants are the best candidates for studies based on genotype and allele frequency, as well as linkage disequilibrium (LD) in founder populations.

METHODS

Six polymorphisms in five folate metabolism‐related genes (MTHFR, MTHFD, MTRR, GCP2, and RFC1) were genotyped on a collection of 1064 DNA samples from populations around the world, which were made available by the Centre d'Étude du Polymorphisme Humain (CEPH) consortium for analysis.

RESULTS

In this study we report the genotype frequencies for variants in the MTHFR, MTHFD, MTRR, GCP2, and RFC1 genes, and the LD for two variants (C677T and A1298C) in MTHFR.

CONCLUSIONS

The rare allele frequency for each of the five genes studied varied widely. LD is strongest in Pakistani and Brazilian populations (D′ = 1.0) and weakest in Mexican populations (D′ = 0.45). These findings will allow the selection of variants that will provide the most power in studies of folate pathway genes involving different ancestral populations, and contribute to our knowledge of the population distribution of selected nutritional gene variants. Birth Defects Research (Part A), 2003. © 2003 Wiley‐Liss, Inc.

     

Sequence variability of <Emphasis Type="Italic">Campylobacter</Emphasis>temperate bacteriophages

Background  

Prophages integrated within the chromosomes of Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found.     

Haplotype-specific <Emphasis Type="Italic">MAPT</Emphasis> exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Background

Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.

Methods

In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.

Results

Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.

Conclusions

We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

     

Intraspecific divergence in sperm morphology of the green sea urchin, <Emphasis Type="Italic">Strongylocentrotus droebachiensis</Emphasis>: implications for selection in broadcast spawners

Background  

Sperm morphology can be highly variable among species, but less is known about patterns of population differentiation within species. Most studies of sperm morphometric variation are done in species with internal fertilization, where sexual selection can be mediated by complex mating behavior and the environment of the female reproductive tract. Far less is known about patterns of sperm evolution in broadcast spawners, where reproductive dynamics are largely carried out at the gametic level. We investigated variation in sperm morphology of a broadcast spawner, the green sea urchin (Strongylocentrotus droebachiensis), within and among spawnings of an individual, among individuals within a population, and among populations. We also examined population-level variation between two reproductive seasons for one population. We then compared among-population quantitative genetic divergence (Q _ST) for sperm characters to divergence at neutral microsatellite markers (F _ST).     

TAS2R38 and Its Influence on Smoking Behavior and Glucose Homeostasis in the German Sorbs

Background

Genetic variants within the bitter taste receptor gene TAS2R38 are associated with sensitivity to bitter taste and are related to eating behavior in the Amish population. Sensitivity to bitter taste is further related to anthropometric traits in an genetically isolated Italian population. We tested whether the TAS2R38 variants (rs713598; rs1726866 and rs10246939) may be related to eating behavior, anthropometric parameters, metabolic traits and consumer goods intake in the German Sorbs.

Materials and Methods

The three SNPs were genotyped in a total cohort of 1007 individuals (male/female: 405/602). The German version of the three-factor eating questionnaire was completed by 548 individuals. Genetic association analyses for smoking behavior, alcohol and coffee intake, eating behavior factors (restraint, disinhibition and hunger) and other metabolic traits were analyzed. Further, by combining the three SNPs we applied comparative haplotype analyses categorizing PAV (proline-alanine-valine) carriers (tasters) vs. homozygous AVI (alanin-valine-isoleucine) carriers (non-tasters).

Results

Significant associations of genetic variants within TAS2R38 were identified with percentage of body fat, which were driven by associations in women. In men, we observed significant associations with 30 min plasma glucose, and area under the curve for plasma glucose (0–120 min) (all adjusted P≤0.05). Further, we found that carriers of at least one PAV allele show significantly lower cigarette smoking per day (P = 0.002) as well as, albeit non-significant, lower alcohol intake. We did not confirm previously reported associations between genetic variants of TAS2R38 and eating behavior.

Conclusion

Our data suggest that genetic variation in TAS2R38 is related to individual body composition measures and may further influence consumer goods intake in the Sorbs possibly via individual sensitivity to bitter taste.     

<Emphasis Type="Italic">Gen</Emphasis><Superscript><Emphasis Type="Italic">Htr</Emphasis></Superscript>: a tool for comparative assessment of genetic heterogeneity in microbial genomes generated by massive short-read sequencing

Background  

Microevolution is the study of short-term changes of alleles within a population and their effects on the phenotype of organisms. The result of the below-species-level evolution is heterogeneity, where populations consist of subpopulations with a large number of structural variations. Heterogeneity analysis is thus essential to our understanding of how selective and neutral forces shape bacterial populations over a short period of time. The Solexa Genome Analyzer, a next-generation sequencing platform, allows millions of short sequencing reads to be obtained with great accuracy, allowing for the ability to study the dynamics of the bacterial population at the whole genome level. The tool referred to as Gen ^Htr was developed for genome-wide heterogeneity analysis.     

Gene expression variation in African and European populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

Differences in levels of gene expression among individuals are an important source of phenotypic variation within populations. Recent microarray studies have revealed that expression variation is abundant in many species, including Drosophila melanogaster. However, previous expression surveys in this species generally focused on a small number of laboratory strains established from derived populations. Thus, these studies were not ideal for population genetic analyses.     

Recombination,cryptic clades and neutral molecular divergence of the microcystin synthetase (<Emphasis Type="Italic">mcy</Emphasis>) genes of toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Background  

The water-bloom-forming cyanobacterium Microcystis aeruginosa is a known producer of various kinds of toxic and bioactive chemicals. Of these, hepatotoxic cyclic heptapeptides microcystins have been studied most intensively due to increasing concerns for human health risks and environmental damage. More than 70 variants of microcystins are known, and a single microcystin synthetase (mcy) gene cluster consisting of 10 genes (mcyA to mcyJ) has been identified to be responsible for the production of all known variants of microcystins. Our previous multilocus sequence typing (MLST) analysis of the seven housekeeping genes indicated that microcystin-producing strains of M. aeruginosa are classified into two phylogenetic groups.     

The missense variation landscape of FTO,MC4R,and TMEM18 in obese children of African Ancestry

Objective:

Common variation at the loci harboring fat mass and obesity (FTO), melanocortin receptor 4 (MC4R), and transmembrane protein 18 (TMEM18) is consistently reported as being statistically most strongly associated with obesity. Investigations if these loci also harbor rarer missense variants that confer substantially higher risk of common childhood obesity in African American (AA) children were conducted.

Design and Methods:

The exons of FTO, MC4R, and TMEM18 in an initial subset of our cohort were sequenced, that is, 200 obese (BMI≥95th percentile) and 200 lean AA children (BMI≤5th percentile). Any missense exonic variants that were uncovered went on to be further genotyped in a further 768 obese and 768 lean (BMI≤50th percentile) children of the same ethnicity.

Results:

A number of exonic variants were observed from our sequencing effort: seven in FTO, of which four were non‐synonymous (A163T, G182A, M400V, and A405V), thirteen in MC4R, of which six were non‐synonymous (V103I, N123S, S136A, F202L, N240S, and I251L), and four in TMEM18, of which two were non‐synonymous (P2S and V113L). Follow‐up genotyping of these missense variants revealed only one significant difference in allele frequency between cases and controls, namely with N240S in MC4R (Fisher's exact P = 0.0001).

Conclusion:

In summary, moderately rare missense variants within the FTO, MC4R, and TMEM18 genes observed in our study did not confer risk of common childhood obesity in African Americans except for a degree of evidence for one known loss‐of‐function variant in MC4R.     

Molecular Basis of the Size Polymorphism of the First Intron of the<Emphasis Type="Italic">Adh-1</Emphasis> Gene of the Mediterranean Fruit Fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The first intron of the gene encoding one of the alcohol dehydrogenase isoenzymes (ADH-1) in Ceratitis capitata is highly polymorphic in size. Five size variants of this intron were isolated from different strains and populations and characterized. Restriction map and sequence analysis showed that the intron size polymorphism is due to the presence or absence of (a) a copy of a defective mariner-like element, postdoc; (b) an 550-bp 3 indel which exhibits no similarity to any known sequence; and (c) a central duplication of 704 bp consisting of part of the 3 end of the postdoc element, the region between postdoc and the 3 indel, and the first 20 bp of the 3 indel. The homologous Adh-1 intron was amplified from the congeneric species, Ceratitis rosa, in order to obtain an outgroup for comparative and phylogenetic analyses. The C. rosa introns were polymorphic in size, ranging from about 1100 to 2000 bp, the major difference between them being the presence or absence of a mariner-like element Crmar2, unrelated to the postdoc element. Phylogenetic analysis suggests that the shorter intron variants in C. capitata may represent the ancestral form of the intron, the longest variants apparently being the most recent.     

Superinfection occurs in <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> infected sheep irrespective of infection phase and protection status

Background  

Anaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.     

Discovery,localization, and sequence characterization of molecular markers for the crown rust resistance genes <Emphasis Type="Italic">Pc38, Pc39</Emphasis>, and <Emphasis Type="Italic">Pc48</Emphasis> in cultivated oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Molecular markers for the crown rust resistance genes Pc38, Pc39, and Pc48 in cultivated oat (Avena sativa L.) were identified using near-isogenic lines and bulked segregant analysis. Six markers for Pc48, the closest being 6 cM away, were found in a Pendek-39 × Pendek-48 (Pendek3948) population, but none was found in a Pendek-48 × Pendek-38 (Pendek4838) population. Three markers for Pc39 were found in the Pendek3948 population, one of which cosegregated with the gene. This same marker was found to be 6 cM away from the gene in an OT328 × Dumont (OT328Du) population. Nine markers for Pc38 were found in the Pendek4838 population, eight of which are within 2 cM of the gene. One other marker for Pc38 was found in the OT328Du population; however, comparative mapping suggests that the Pc38 region in OT328Du is in a different location than that in Pendek4838. A number of markers unlinked to the genes under study formed linkage groups in both the Pendek3948 and Pendek4838 populations. Four of these show homology or homoeology to each other and to the Pc39 region in Pendek3948. Two RFLP clones closely linked to Pc38 code for a putative leucine-rich repeat transmembrane protein kinase and a cre3 resistance gene analogue. This study provides information to support molecular breeding in oat, and contributes to ongoing research into genomic regions associated with fungal pathogen resistance.     

vFitness: a web-based computing tool for improving estimation of <Emphasis Type="Italic">in vitro</Emphasis> HIV-1 fitness experiments

Background  

The replication rate (or fitness) between viral variants has been investigated in vivo and in vitro for human immunodeficiency virus (HIV). HIV fitness plays an important role in the development and persistence of drug resistance. The accurate estimation of viral fitness relies on complicated computations based on statistical methods. This calls for tools that are easy to access and intuitive to use for various experiments of viral fitness.     

Polymorphism,genetic exchange and intragenic recombination of the aureolysin gene among <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>strains

Background  

Staphylococcus aureus expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of S. aureus, relatively little information was so far available concerning the aur gene diversity and mobility within and between the major subdivisions of the S. aureus population. Therefore, an epidemiologically and genetically diverse collection of S. aureus strains was used to determine the range of aureolysin (aur) gene polymorphism.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> sigma B-dependent emergence of small-colony variants and biofilm production following exposure to <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> 4-hydroxy-2-heptylquinoline-<Emphasis Type="Italic">N-</Emphasis> oxide

Background  

Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.     

Distinguishing importation from diversification of quinolone-resistant <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> by molecular evolutionary analysis

Background  

Distinguishing the recent introduction of quinolone resistant gonococci into a population from diversification of resistant strains already in the population is important for planning effective infection control strategies. We applied molecular evolutionary analyses to DNA sequences from 9 housekeeping genes and gyrA, parC and porB of 24 quinolone resistant N. gonorrhoeae (QRNG) and 24 quinolone sensitive isolates collected in Israel during 2000–2001.