The Role of Fusion in Ant Chromosome Evolution: Insights from Cytogenetic Analysis Using a Molecular Phylogenetic Approach in the Genus Mycetophylax

Among insect taxa, ants exhibit one of the most variable chromosome numbers ranging from n = 1 to n = 60. This high karyotype diversity is suggested to be correlated to ants diversification. The karyotype evolution of ants is usually understood in terms of Robertsonian rearrangements towards an increase in chromosome numbers. The ant genus Mycetophylax is a small monogynous basal Attini ant (Formicidae: Myrmicinae), endemic to sand dunes along the Brazilian coastlines. A recent taxonomic revision validates three species, Mycetophylax morschi, M. conformis and M. simplex. In this paper, we cytogenetically characterized all species that belongs to the genus and analyzed the karyotypic evolution of Mycetophylax in the context of a molecular phylogeny and ancestral character state reconstruction. M. morschi showed a polymorphic number of chromosomes, with colonies showing 2n = 26 and 2n = 30 chromosomes. M. conformis presented a diploid chromosome number of 30 chromosomes, while M. simplex showed 36 chromosomes. The probabilistic models suggest that the ancestral haploid chromosome number of Mycetophylax was 17 (Likelihood framework) or 18 (Bayesian framework). The analysis also suggested that fusions were responsible for the evolutionary reduction in chromosome numbers of M. conformis and M. morschi karyotypes whereas fission may determines the M. simplex karyotype. These results obtained show the importance of fusions in chromosome changes towards a chromosome number reduction in Formicidae and how a phylogenetic background can be used to reconstruct hypotheses about chromosomes evolution.     

Chromosome Painting in Three Species of Buteoninae: A Cytogenetic Signature Reinforces the Monophyly of South American Species

Buteoninae (Falconiformes, Accipitridae) consist of the widely distributed genus Buteo, and several closely related species in a group called “sub-buteonine hawks”, such as Buteogallus, Parabuteo, Asturina, Leucopternis and Busarellus, with unsolved phylogenetic relationships. Diploid number ranges between 2n = 66 and 2n = 68. Only one species, L. albicollis had its karyotype analyzed by molecular cytogenetics. The aim of this study was to present chromosomal analysis of three species of Buteoninae: Rupornis magnirostris, Asturina nitida and Buteogallus meridionallis using fluorescence in situ hybridization (FISH) experiments with telomeric and rDNA probes, as well as whole chromosome probes derived from Gallus gallus and Leucopternis albicollis. The three species analyzed herein showed similar karyotypes, with 2n = 68. Telomeric probes showed some interstitial telomeric sequences, which could be resulted by fusion processes occurred in the chromosomal evolution of the group, including the one found in the tassociation GGA1p/GGA6. In fact, this association was observed in all the three species analyzed in this paper, and also in L. albicollis, suggesting that it represents a cytogenetic signature which reinforces the monophyly of Neotropical buteoninae species.     

Archolaemus janeae (Gymnotiformes,Teleostei): First insights into karyotype and repetitive DNA distribution in two populations of the Amazon

Archolaemus, one of the five genera of Neotropical freshwater fish of the family Sternopygidae (Gymnotiformes), was long considered a monotypic genus represented by Archolaemus blax. Currently, it consists of six species, most of them occurring in the Amazon region. There are no cytogenetic data for species of this genus. In the present study, we used classical cytogenetics (conventional staining and C‐banding) and molecular cytogenetics (probes of telomeric sequences and multigenic families 18S rDNA, 5S rDNA, and U2 snDNA) to study the karyotype of Archolaemus janeae from Xingu and Tapajós rivers in the state of Pará (Brazil). The results showed that the two populations have identical karyotypes with 46 chromosomes: four submetacentric and 42 acrocentric (2n = 46; 4m/sm + 42a). Constitutive heterochromatin occurs in the centromeric region of all chromosomes, in addition to small bands in the interstitial and distal regions of some pairs. The 18S rDNA occurs in the distal region of the short arm of pair 2; the 5S rDNA occurs in five chromosome pairs; and the U2 snDNA sequence occurs in chromosome pairs 3, 6, and 13. No interstitial telomeric sequence was observed. These results show karyotypic similarity between the studied populations suggesting the existence of a single species and are of great importance as a reference for future cytotaxonomic studies of the genus.     

Reconstructing the Evolution of Brachypodium Genomes Using Comparative Chromosome Painting

Brachypodium distachyon is a model for the temperate cereals and grasses and has a biology, genomics infrastructure and cytogenetic platform fit for purpose. It is a member of a genus with fewer than 20 species, which have different genome sizes, basic chromosome numbers and ploidy levels. The phylogeny and interspecific relationships of this group have not to date been resolved by sequence comparisons and karyotypical studies. The aims of this study are not only to reconstruct the evolution of Brachypodium karyotypes to resolve the phylogeny, but also to highlight the mechanisms that shape the evolution of grass genomes. This was achieved through the use of comparative chromosome painting (CCP) which hybridises fluorescent, chromosome-specific probes derived from B. distachyon to homoeologous meiotic chromosomes of its close relatives. The study included five diploids (B. distachyon 2n = 10, B. sylvaticum 2n = 18, B. pinnatum 2n = 16; 2n = 18, B. arbuscula 2n = 18 and B. stacei 2n = 20) three allotetraploids (B. pinnatum 2n = 28, B. phoenicoides 2n = 28 and B. hybridum 2n = 30), and two species of unknown ploidy (B. retusum 2n = 38 and B. mexicanum 2n = 40). On the basis of the patterns of hybridisation and incorporating published data, we propose two alternative, but similar, models of karyotype evolution in the genus Brachypodium. According to the first model, the extant genome of B. distachyon derives from B. mexicanum or B. stacei by several rounds of descending dysploidy, and the other diploids evolve from B. distachyon via ascending dysploidy. The allotetraploids arise by interspecific hybridisation and chromosome doubling between B. distachyon and other diploids. The second model differs from the first insofar as it incorporates an intermediate 2n = 18 species between the B. mexicanum or B. stacei progenitors and the dysploidic B. distachyon.     

Sex Chromosomes and Karyotype of the (Nearly) Mythical Creature,the Gila Monster,Heloderma suspectum (Squamata: Helodermatidae)

A wide variety of sex determination systems exist among squamate reptiles. They can therefore serve as an important model for studies of evolutionary transitions among particular sex determination systems. However, we still have only a limited knowledge of sex determination in certain important lineages of squamates. In this respect, one of the most understudied groups is the family Helodermatidae (Anguimorpha) encompassing the only two venomous species of lizards which are potentially lethal to human beings. We uncovered homomorphic ZZ/ZW sex chromosomes in the Gila monster (Heloderma suspectum) with a highly heterochromatic W chromosome. The sex chromosomes are morphologically similar to the ZZ/ZW sex chromosomes of monitor lizards (Varanidae). If the sex chromosomes of helodermatids and varanids are homologous, female heterogamety may be ancestral for the whole Anguimorpha group. Moreover, we found that the karyotype of the Gila monster consists of 2n = 36 chromosomes (14 larger metacentric chromosomes and 22 acrocentric microchromosomes). 2n = 36 is the widely distributed chromosomal number among squamates. In his pioneering works representing the only previous cytogenetic examination of the family Helodermatidae, Matthey reported the karyotype as 2n = 38 and suggested a different chromosomal morphology for this species. We believe that this was probably erroneously. We also discovered a strong accumulation of telomeric sequences on several pairs of microchromosomes in the Gila monster, which is a trait documented relatively rarely in vertebrates. These new data fill an important gap in our understanding of the sex determination and karyotype evolution of squamates.     

DNA barcode detects high genetic structure within neotropical bird species

Background

Towards lower latitudes the number of recognized species is not only higher, but also phylogeographic subdivision within species is more pronounced. Moreover, new genetically isolated populations are often described in recent phylogenies of Neotropical birds suggesting that the number of species in the region is underestimated. Previous COI barcoding of Argentinean bird species showed more complex patterns of regional divergence in the Neotropical than in the North American avifauna.

Methods and Findings

Here we analyzed 1,431 samples from 561 different species to extend the Neotropical bird barcode survey to lower latitudes, and detected even higher geographic structure within species than reported previously. About 93% (520) of the species were identified correctly from their DNA barcodes. The remaining 41 species were not monophyletic in their COI sequences because they shared barcode sequences with closely related species (N = 21) or contained very divergent clusters suggestive of putative new species embedded within the gene tree (N = 20). Deep intraspecific divergences overlapping with among-species differences were detected in 48 species, often with samples from large geographic areas and several including multiple subspecies. This strong population genetic structure often coincided with breaks between different ecoregions or areas of endemism.

Conclusions

The taxonomic uncertainty associated with the high incidence of non-monophyletic species and discovery of putative species obscures studies of historical patterns of species diversification in the Neotropical region. We showed that COI barcodes are a valuable tool to indicate which taxa would benefit from more extensive taxonomic revisions with multilocus approaches. Moreover, our results support hypotheses that the megadiversity of birds in the region is associated with multiple geographic processes starting well before the Quaternary and extending to more recent geological periods.     

Study of four Neotropical species of tree crickets Oecanthus Serville, 1831 (Orthoptera,Gryllidae) using cytogenetic and molecular markers

Karyotypes in the worldwide subfamily Oecanthinae show variations in diploid number, chromosome morphology, and sex-chromosome system. This study described the chromosome set and phylogenetic relationships of four Neotropical species, Oecanthus lineolatus, O. valensis, O. pallidus, and O. pictus. We used classical cytogenetics and Bayesian Inference for phylogenetic reconstruction, using the mitochondrial genes COI, 12S rRNA, and 16S rRNA; and analyzed the phylogenetic patterns of changes in chromosome numbers, using ChromEvol. We observed differences in chromosome number among species and two different sex-chromosome systems. Oecanthus pictus showed 2n = 21, X0♂/22, XX♀; O. lineolatus, 2n = 20, XY♂/XX♀; and O. valensis and O. pallidus, 2n = 18, XY♂/XX♀. The karyotype of Oecanthus was asymmetric, one group with large chromosomes and variation in heterochromatin distribution, and another with small acrocentric chromosomes. The phylogenetic tree recovered two main groups: one with the Palearctic species and another with species from different bioregions, but with low posterior probability. The Neotropical species grouped separately, O. valensis and O. pictus with Nearctic and Ethiopian species, and O. pallidus and O. lineolatus in another, well-supported clade. Together, the phylogenic and chromosome data suggest descending dysploidy events during the evolution of the group. Keywords: Insect, karyotype, Chromosome, Bayesian Inference, Oecanthinae     

Ixeridium calcicola (Compositae), a New Limestone Endemic from Taiwan,with Notes on Its Atypical Basic Chromosome Number,Phylogenetic Affinities,and a Limestone Refugium Hypothesis

A new species Ixeridium calcicola (Compositae) endemic to middle altitude (ca 1,000–2,000 m asl) limestone mountains of eastcentral Taiwan is described based on morphological and chromosome cytological observations and molecular phylogenetic analyses. Ixeridium calcicola resembles Ixeridium transnokoense, endemic to upper montane and alpine ranges (2,600–3,500 m asl) of Taiwan, in the dwarf habit, but differs in the oblong to lanceolate leaf blades (vs. linear to linear-lanceolate), the presence of mucronulate teeth on the leaf margin and petiole (vs. smooth to very sparse), the dark purple lower leaf surface (vs. greenish), the capitulum with 10 to 12 florets (vs. 5 to 7) and 8 to 10 inner phyllaries (vs. 5, rarely to 7). The basic chromosome number in Ixeridium was known as X = 7. However, the new species has a basic chromosome number of X = 8, as recorded also in the closely related Ixeris. Molecular phylogenetic analyses with the expanded sampling of Ixeridium and Ixeris including both type species supported the monophyly of each of the genera and the placement of the new species in Ixeridium. The result of the phylogenetic analyses and detailed observation of the chromosome morphology revealed that X = 8 in Ixeridium calcicola is derived from centric fission in an ancestral karyomorphotype with X = 7 in Ixeridium. Ixeridium calcicola and Ixeridium transnokoense formed a Taiwan endemic lineage and their estimated divergence time was in the middle Pleistocene. Their common ancestral lineage may have experienced altitudinal distribution shifts in response to glacial-interglacial temperature fluctuation, and a lineage which had not retreated to alpine ranges in an interglacial period likely survived in a limestone refugium, where ordinary plant species did not grow, leading to allopatric speciation.     

Comparative analysis of rDNA location in five Neotropical gomphocerine grasshopper species

We report here, for the first time, the chromosome complement, number and location of the nucleolar organizer regions (NORs) revealed by silver staining (AgNO₃) and fluorescent in situ hybridization (FISH) in five Neotropical gomphocerine species: Rhammatocerus brasiliensis, R. brunneri, R. palustris, R. pictus and Amblytropidia sp. The objective of this study was to summarize available data and propose a model of chromosome evolution in Neotropical gomphocerines. All five species studied showed chromosome numbers consisting of 2n = 23,X0 in males and 2n = 24,XX in females. Amblytropidia sp. was the only species showing a bivalent (M₈) with megameric behavior during meiosis. The rDNA sites were restricted to autosomal pairs, i.e. the pericentromeric region of the S₉ chromosome, the consensus NOR location in all five species. R. brasiliensis was the only species showing additional NORs on M₄ and M₆ pairs which, likewise the S₉ NOR, were active in all cells analyzed. Comparison of these results with those reported previously in Palearctic gomphocerine species suggests higher resemblance of Neotropical species with the Old World species also possessing 23/24 chromosomes. Evolutionary mechanisms responsible for the observed interspecific variation in NOR location in this group are discussed.     

Genome-Wide Association Study of Lp-PLA2 Activity and Mass in the Framingham Heart Study

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is an emerging risk factor and therapeutic target for cardiovascular disease. The activity and mass of this enzyme are heritable traits, but major genetic determinants have not been explored in a systematic, genome-wide fashion. We carried out a genome-wide association study of Lp-PLA₂ activity and mass in 6,668 Caucasian subjects from the population-based Framingham Heart Study. Clinical data and genotypes from the Affymetrix 550K SNP array were obtained from the open-access Framingham SHARe project. Each polymorphism that passed quality control was tested for associations with Lp-PLA₂ activity and mass using linear mixed models implemented in the R statistical package, accounting for familial correlations, and controlling for age, sex, smoking, lipid-lowering-medication use, and cohort. For Lp-PLA₂ activity, polymorphisms at four independent loci reached genome-wide significance, including the APOE/APOC1 region on chromosome 19 (p = 6×10⁻²⁴); CELSR2/PSRC1 on chromosome 1 (p = 3×10⁻¹⁵); SCARB1 on chromosome 12 (p = 1×10⁻⁸) and ZNF259/BUD13 in the APOA5/APOA1 gene region on chromosome 11 (p = 4×10⁻⁸). All of these remained significant after accounting for associations with LDL cholesterol, HDL cholesterol, or triglycerides. For Lp-PLA₂ mass, 12 SNPs achieved genome-wide significance, all clustering in a region on chromosome 6p12.3 near the PLA2G7 gene. Our analyses demonstrate that genetic polymorphisms may contribute to inter-individual variation in Lp-PLA₂ activity and mass.     

The Role of Historical Barriers in the Diversification Processes in Open Vegetation Formations during the Miocene/Pliocene Using an Ancient Rodent Lineage as a Model

The Neotropics harbors a high diversity of species and several hypotheses have been proposed to account for this pattern. However, while species of forested domains are frequently studied, less is known of species from open vegetation formations occupying, altogether, a larger area than the Amazon Forest. Here we evaluate the role of historical barriers and the riverine hypothesis in the speciation patterns of small mammals by analyzing an ancient rodent lineage (Thrichomys, Hystricomorpha). Phylogenetic and biogeographic analyses were carried out with mitochondrial and nuclear DNA markers to analyze the evolutionary relationships between Thrichomys lineages occurring in dry domains along both banks of the Rio São Francisco. This river is one of the longest of South America whose course and water flow have been modified by inland tectonic activities and climate changes. Molecular data showed a higher number of lineages than previously described. The T. inermis species complex with 2n = 26, FN = 48 was observed in both banks of the river showing a paraphyletic arrangement, suggesting that river crossing had occurred, from east to west. A similar pattern was also observed for the T. apereoides complex. Thrichomys speciation occurred in Late Miocene when the river followed a different course. The current geographic distribution of Thrichomys species and their phylogenetic relationships suggested the existence of frequent past connections between both banks in the middle section of the Rio São Francisco. The extensive palaeodune region found in this area has been identified as a centre of endemism of several vertebrate species and is likely to be a center of Thrichomys diversification.     

Apparent Survival Rates of Forest Birds in Eastern Ecuador Revisited: Improvement in Precision but No Change in Estimates

Knowledge of survival rates of Neotropical landbirds remains limited, with estimates of apparent survival available from relatively few sites and species. Previously, capture-mark-recapture models were used to estimate apparent survival of 31 species (30 passerines, 1 Trochilidae) from eastern Ecuador based on data collected from 2001 to 2006. Here, estimates are updated with data from 2001-2012 to determine how additional years of data affect estimates; estimates for six additional species are provided. Models assuming constant survival had highest support for 19 of 31 species when based on 12 years of data compared to 27 when based on six; models incorporating effects of transients had the highest support for 12 of 31 species compared to four when based on 12 and six years, respectively. Average apparent survival based on the most highly-supported model (based on model averaging, when appropriate) was 0.59 (± 0.02 SE) across 30 species of passerines when based on 12 years and 0.57 (± 0.02) when based on six. Standard errors of survival estimates based on 12 years were approximately half those based on six years. Of 31 species in both data sets, estimates of apparent survival were somewhat lower for 13, somewhat higher for 17, and remained unchanged for one; confidence intervals for estimates based on six and 12 years of data overlapped for all species. Results indicate that estimates of apparent survival are comparable but more precise when based on longer-term data sets; standard error of the estimates was negatively correlated with numbers of captures (r_s = −0.72) and recaptures (r_s = −0.93, P<0.001 in both cases). Thus, reasonable estimates of apparent survival may be obtained with relatively few years of data if sample sizes are sufficient.     

Karyotype, sex determination, and meiotic chromosome behavior in two pholcid (Araneomorphae, Pholcidae) spiders: implications for karyotype evolution

There are 1,111 species of pholcid spiders, of which less than 2% have published karyotypes. Our aim in this study was to determine the karyotypes and sex determination mechanisms of two species of pholcids: Physocyclus mexicanus (Banks, 1898) and Holocnemus pluchei (Scopoli, 1763), and to observe sex chromosome behavior during meiosis. We constructed karyotypes for P. mexicanus and H. pluchei using information from both living and fixed cells. We found that P. mexicanus has a chromosome number of 2n = 15 in males and 2n = 16 in females with X0-XX sex determination, like other members of the genus Physocyclus. H. pluchei has a chromosome number of 2n = 28 in males and 2n = 28 in females with XY-XX sex determination, which is substantially different from its closest relatives. These data contribute to our knowledge of the evolution of this large and geographically ubiquitous family, and are the first evidence of XY-XX sex determination in pholcids.     

Sequential Cross-Species Chromosome Painting among River Buffalo,Cattle, Sheep and Goat: A Useful Tool for Chromosome Abnormalities Diagnosis within the Family Bovidae

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards.     

The Unique hmuY Gene Sequence as a Specific Marker of Porphyromonas gingivalis

Porphyromonas gingivalis, a major etiological agent of chronic periodontitis, acquires heme from host hemoproteins using the HmuY hemophore. The aim of this study was to develop a specific P. gingivalis marker based on a hmuY gene sequence. Subgingival samples were collected from 66 patients with chronic periodontitis and 40 healthy subjects and the entire hmuY gene was analyzed in positive samples. Phylogenetic analyses demonstrated that both the amino acid sequence of the HmuY protein and the nucleotide sequence of the hmuY gene are unique among P. gingivalis strains/isolates and show low identity to sequences found in other species (below 50 and 56%, respectively). In agreement with these findings, a set of hmuY gene-based primers and standard/real-time PCR with SYBR Green chemistry allowed us to specifically detect P. gingivalis in patients with chronic periodontitis (77.3%) and healthy subjects (20%), the latter possessing lower number of P. gingivalis cells and total bacterial cells. Isolates from healthy subjects possess the hmuY gene-based nucleotide sequence pattern occurring in W83/W50/A7436 (n = 4), 381/ATCC 33277 (n = 3) or TDC60 (n = 1) strains, whereas those from patients typically have TDC60 (n = 21), W83/W50/A7436 (n = 17) and 381/ATCC 33277 (n = 13) strains. We observed a significant correlation between periodontal index of risk of infectiousness (PIRI) and the presence/absence of P. gingivalis (regardless of the hmuY gene-based sequence pattern of the isolate identified [r = 0.43; P = 0.0002] and considering particular isolate pattern [r = 0.38; P = 0.0012]). In conclusion, we demonstrated that the hmuY gene sequence or its fragments may be used as one of the molecular markers of P. gingivalis.     

Numerical and structural chromosome variation in the swarm-founding wasp Metapolybia decorata Gribodo 1896 (Hymenoptera, Vespidae)

The Neotropical Polistinae wasps are diverse in taxonomy, social behavior, and nesting founding characteristics. Although some species in this group have been used as models for studies on wasp’s biology, they are poorly known in terms of cytogenetics. Here we reported an intraspecific numerical-structural chromosome variation in the swarm-founding wasp Metapolybia decorata from the Brazilian Atlantic Rainforest using conventional and molecular cytogenetic techniques. The observed structural chromosome change involved a telomeric fusion that resulted in a chromosome number range of 2n = 34–36. The origin and geographic distribution of the variant chromosome forms as well as their frequency and maintenance in the studied populations are discussed. In addition, we reported a novel and geographically restricted deletion in the fused chromosomes indicating that the species is undergoing a continued process of karyotype evolution leading to fused chromosome stabilization by elimination of inactive centromeric sequences. Evidence of differences in the telomeric sequences of this wasp was also found by in situ hybridization using the motif (T₂AG₂)₇ as probe.     

The Association between DNA Copy Number Aberrations at Chromosome 5q22 and Gastric Cancer

Background

Gastric cancer is common cancer. Discovering novel genetic biomarkers might help to identify high-risk individuals. Copy number variation (CNV) has recently been shown to influence risk for several cancers. The aim of the present study was sought to test the association between copy number at a variant region and GC.

Methods

A total of 110 gastric cancer patients and 325 healthy volunteers were enrolled in this study. We searched for a CNV and found a CNV (Variation 7468) containing part of the APC gene, the SRP19 gene and the REEP5 gene. We chose four probes targeting at APC-intron8, APC-exon9, SRP19 and REEP5 to interrogate this CNV. Specific Taqman probes labeled by different reporter fluorophores were used in a real-time PCR platform to obtain copy number. Both the original non-integer data and transformed integer data on copy number were used for analyses.

Results

Gastric caner patients had a lower non-integer copy number than controls for the APC-exon9 probe (Adjusted p = 0.026) and SRP19 probe (Adjusted p = 0.002). The analysis of integer copy number yielded a similar pattern although less significant (Adjusted p = 0.07 for APC-exon9 probe and Adjusted p = 0.02 for SRP19 probe).

Conclusions

Losses of a CNV at 5q22, especially in the DNA region surrounding APC-exon 9, may be associated with a higher risk of gastric cancer.     

Screening for EGFR Amplifications with a Novel Method and Their Significance for the Outcome of Glioblastoma Patients

Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (i.e. TP53 and IDH1 mutations, CDKN2A deletion, EGFR amplification, chromosome 7 polysomy and EGFRvIII expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for EGFR amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient’s age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, EGFR amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, EGFR amplification was related to a better prognosis in combination with the homozygous CDKN2A deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased EGFR gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient’s tumour molecular characteristics in the selection of the therapy.     

Mitochondrial Control Region Alterations and Breast Cancer Risk: A Study in South Indian Population

Background

Mitochondrial displacement loop (D-loop) is the hot spot for mitochondrial DNA (mtDNA) alterations which influence the generation of cellular reactive oxygen species (ROS). Association of D-loop alterations with breast cancer has been reported in few ethnic groups; however none of the reports were documented from Indian subcontinent.

Methodology

We screened the entire mitochondrial D-loop region (1124 bp) of breast cancer patients (n = 213) and controls (n = 207) of south Indian origin by PCR-sequencing analysis. Haplotype frequencies for significant loci, the standardized disequilibrium coefficient (D′) for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software.

Principal Findings

We identified 7 novel mutations and 170 reported polymorphisms in the D-loop region of patients and/or controls. Polymorphisms were predominantly located in hypervariable region I (60%) than in II (30%) of D-loop region. The frequencies of 310‘C’ insertion (P = 0.018), T16189C (P = 0.0019) variants and 310‘C’ins/16189C (P = 0.00019) haplotype were significantly higher in cases than in controls. Furthermore, strong LD was observed between nucleotide position 310 and 16189 in controls (D′ = 0.49) as compared to patients (D′ = 0.14).

Conclusions

Mitochondrial D-loop alterations may constitute inherent risk factors for breast cancer development. The analysis of genetic alterations in the D-loop region might help to identify patients at high risk for bad progression, thereby helping to refine therapeutic decisions in breast cancer.     

Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of Dasypyrum villosum L. (H. villosa) by a Single DNA Probe

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.