Segmental dataset and whole body expression data do not support the hypothesis that non-random movement is an intrinsic property of Drosophila retrogenes

Background: Several studies in Drosophila have shown excessive movement of retrogenes from the X chromosome to autosomes, and that these genes are frequently expressed in the testis. This phenomenon has led to several hypotheses invoking natural selection as the process driving male-biased genes to the autosomes. Metta and Schlotterer (BMC Evol Biol 2010, 10:114) analyzed a set of retrogenes where the parental gene has been subsequently lost. They assumed that this class of retrogenes replaced the ancestral functions of the parental gene, and reported that these retrogenes, although mostly originating from movement out of the X chromosome, showed female-biased or unbiased expression. These observations led the authors to suggest that selective forces (such as meiotic sex chromosome inactivation and sexual antagonism) were not responsible for the observed pattern of retrogene movement out of the X chromosome. Results: We reanalyzed the dataset published by Metta and Schlotterer and found several issues that led us to a different conclusion. In particular, Metta and Schlotterer used a dataset combined with expression data in which significant sex-biased expression is not detectable. First, the authors used a segmental dataset where the genes selected for analysis were less testis-biased in expression than those that were excluded from the study. Second, sex-biased expression was defined by comparing male and female whole-body data and not the expression of these genes in gonadal tissues. This approach significantly reduces the probability of detecting sex-biased expressed genes, which explains why the vast majority of the genes analyzed (parental and retrogenes) were equally expressed in both males and females. Third, the female-biased expression observed by Metta and Schltterer is mostly found for parental genes located on the X chromosome, which is known to be enriched with genes with female-biased expression. Fourth, using additional gonad expression data, we found that autosomal genes analyzed by Metta and Schlotterer are less up regulated in ovaries and have higher chance to be expressed in meiotic cells of spermatogenesis when compared to X-linked genes. Conclusions: The criteria used to select retrogenes and the sex-biased expression data based on whole adult flies generated a segmental dataset of female-biased and unbiased expressed genes that was unable to detect the higher propensity of autosomal retrogenes to be expressed in males. Thus, there is no support for the authors' view that the movement of new retrogenes, which originated from X-linked parental genes, was not driven by selection. Therefore, selection-based genetic models remain the most parsimonious explanations for the observed chromosomal distribution of retrogenes.

Chromosome painting in three-toed sloths: a cytogenetic signature and ancestral karyotype for Xenarthra

Background: Xenarthra (sloths, armadillos and anteaters) represent one of four currently recognized Eutherian mammal supraorders. Some phylogenomic studies point to the possibility of Xenarthra being at the base of the Eutherian tree, together or not with the supraorder Afrotheria. We performed painting with human autosomes and X-chromosome specific probes on metaphases of two three-toed sloths: Bradypus torquatus and B. variegatus. These species represent the fourth of the five extant Xenarthra families to be studied with this approach. Results: Eleven human chromosomes were conserved as one block in both B. torquatus and B. variegatus: (HSA 5, 6, 9, 11, 13, 14, 15, 17, 18, 20, 21 and the X chromosome). B. torquatus, three additional human chromosomes were conserved intact (HSA 1, 3 and 4). The remaining human chromosomes were represented by two or three segments on each sloth. Seven associations between human chromosomes were detected in the karyotypes of both B. torquatus and B. variegatus: HSA 3/21, 4/8, 7/10, 7/16, 12/22, 14/15 and 17/19. The ancestral Eutherian association 16/19 was not detected in the Bradypus species. Conclusions: Our results together with previous reports enabled us to propose a hypothetical ancestral Xenarthran karyotype with 48 chromosomes that would differ from the proposed ancestral Eutherian karyotype by the presence of the association HSA 7/10 and by the split of HSA 8 into three blocks, instead of the two found in the Eutherian ancestor. These same chromosome features point to the monophyly of Xenarthra, making this the second supraorder of placental mammals to have a chromosome signature supporting its monophyly.

Variable patterns of Y chromosome homology in Akodontini rodents (Sigmodontinae): a phylogenetic signal revealed by chromosome painting

The Akodontini is the second most speciose tribe of sigmodontine rodents, one of the most diverse groups of neotropical mammals. Molecular phylogenetic analyses are discordant regarding the interrelationships of genera, with low support for some clades. However, two clades are concordant, one (clade A) with Akodon sensu strictu (excluding Akodon serrensis), "Akodon" serrensis, Bibimys, Deltamys, Juscelinomys, Necromys, Oxymycterus, Podoxymys, Thalpomys and Thaptomys, and another (clade B) with Blarinomys, Brucepattersonius, Kunsia, Lenoxus and Scapteromys. Here, we present chromosome painting using Akodon paranaensis (APA) Y paint, after suppression of simple repetitive sequences, on ten Akodontini genera. Partial Y chromosome homology, in addition to the homology already reported on the Akodon genus, was detected on the Y chromosomes of "A." serrensis, Thaptomys, Deltamys, Necromys and Thalpomys and on Y and X chromosomes in Oxymycterus. In Blarinomys, Brucepattersonius, Scapteromys and Kunsia, no APA Y signal was observed using different hybridization conditions; APA X paint gave positive signals only on the X chromosome in all genera. The Y chromosome homology was variable in size and positioning among the species studied as follow: (1) whole acrocentric Y chromosome in Akodon and "A." serrensis, (2) Yp and pericentromeric region in submetacentric Y of Necromys and Thaptomys, (3) pericentromeric region in acrocentric Y of Deltamys, (4) distal Yq in the acrocentric Y chromosome of Thalpomys and (5) proximal Yq in the acrocentric Y and Xp in the basal clade A genus Oxymycterus. The results suggest that the homology involves pairing (pseudoautosomal) and additional regions that have undergone rearrangement during divergence. The widespread Y homology represents a phylogenetic signal in Akodontini that provides additional evidence supporting the monophyly of clade A. The findings also raise questions about the evolution of the pseudoautosomal region observed in Oxymycterus. The Y chromosomes of these closely related species seem to have undergone dynamic rearrangements, including restructuring and reduction of homologous segments. Furthermore, the changes observed may indicate progressive attrition of the Y chromosome in more distantly related species.

A new karyotype for the spiny rat Clyomys laticeps (Thomas, 1909) (Rodentia, Echimyidae) from Central Brazil

Clyomys Thomas, 1916 is a semifossorial rodent genus of spiny rats represented by only one species, C. laticeps, which inhabits the tropical savannas and grasslands of central Brazil and eastern Paraguay. Here we describe a new karyotype of C. laticeps found in populations of Emas National Park, Goias state, Brazil. The four analyzed specimens had a diploid number (2n) of 32 and a fundamental autosome number (FN) of 54. Cytogenetic data include conventional staining, CBG and GTG-banding. The karyotype presents 12 meta/submetacentric pairs (1 to 12) and 3 pairs of acrocentrics (13 to 15) with gradual decrease in size. The X chromosome is a medium submetacentric and the Y is a medium acrocentric. The semifossorial habits together with habitat specificity could have contributed to the karyological variations found on this genus.

The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.

Cytogenetic instability of dental pulp stem cell lines

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

Clinico-genetic aspects of a pediatric non-neurofibromatosis type 1 malignant triton tumor with loss of chromosome X

Malignant triton tumor (MTT) is an aggressive peripheral nerve sheath tumor with rhabdomyoblastic differentiation. Less than 100 cases have been described, being mostly male children with type 1 neurofibromatosis. We report a 6-year-old female with MTT and no diagnostic criteria for neurofibromatosis type 1. Cytogenetic analysis showed a 46,X,-X[4]/46,XX[16] karyotype. She underwent a transfemoral amputation and chemotherapy and is free of disease 15 months after diagnosis. The few cytogenetic studies of MTT described in the literature have been inconclusive. Further cytogenetic analyses are needed to understand the role of chromosome X monosomy in the pathogenesis of this rare tumor. Pediatr Blood Cancer 2012; 59: 13201323. (C) 2012 Wiley Periodicals, Inc.

Mutations, Clinical Findings and Survival Estimates in South American Patients with X-Linked Adrenoleukodystrophy

In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics. Methods: X-ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied. Results: We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p. Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1-12.7) and 24.7 (19.8-29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation. Conclusions: This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.

In Vitro Photodynamic Inactivation of Cryptococcus neoformans Melanized Cells with Chloroaluminum Phthalocyanine Nanoemulsion

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 mu m) and light doses (e.g. 10 J cm-2) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C.neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.

Using Prior Information from the Medical Literature in GWAS of Oral Cancer Identifies Novel Susceptibility Variant on Chromosome 4-the AdAPT Method

Background: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS. Methods: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest - the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer. Results: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5 x 10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found. Conclusion: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config).

A complex chromosomal rearrangement involving chromosomes 2, 5, and X in autism spectrum disorder

Here, we describe a female patient with autism spectrum disorder and dysmorphic features that harbors a complex genetic alteration, involving a de novo balanced translocation t(2;X)(q11;q24), a 5q11 segmental trisomy and a maternally inherited isodisomy on chromosome 5. All the possibly damaging genetic effects of such alterations are discussed. In light of recent findings on ASD genetic causes, the hypothesis that all these alterations might be acting in orchestration and contributing to the phenotype is also considered. (C) 2012 Wiley Periodicals, Inc.

Inactivation of Alicyclobacillus acidoterrestris in orange juice by saponin extracts combined with heat-treatment

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. The inactivation of this bacterium by commercial saponin and saponin purified extract from Sapindus saponaria fruits combined with heat-treatment is described. We investigated heat treatment (87, 90, 95, and 99 degrees C) with incubation time ranging from 0 to 50 min, in both concentrated and reconstituted juice. juices were inoculated with 1.0 x 10(4) CFU/mL of A. acidoterrestris spores for the evaluation of the best temperature for inactivation. For the temperatures of 87, 90, and 95 degrees C counts of cell viability decreased rapidly within the first 10 to 20 min of incubation in both concentrated and reconstituted juices; inactivation at 99 degrees C ensued within 1 and 2 min. Combination of commercial saponin (100 mg/L) with a very short incubation time (1 min) at 99 degrees C showed a reduction of 234 log cycle for concentrated juice A. acidoterrestris spores (1.0 x 10(4) CFU/mL) in the first 24 h of incubation after treatments. The most efficient treatment was reached with 300, 400 or 500 mg/L of purified extract of saponins from S. saponaria after 5 days of incubation in concentrated juice, and after 5 days with 300 and 400 mg/L or 72 h with 500 mg/L in reconstituted juice. Commercial saponin and purified extracts from S. saponaria had similar inactivation power on A. acidoterrestris spores, without significant differences (P>0.05). Therefore, purified extract of saponins can be an alternative for the control of A acidoterrestris in fruit juices. (C) 2012 Elsevier B.V. All rights reserved.

Mechanisms of ring chromosome formation, ring instability and clinical consequences

Background The breakpoints and mechanisms of ring chromosome formation were studied and mapped in 14 patients. Methods Several techniques were performed such as genome-wide array, MLPA (Multiplex Ligation-Dependent Probe Amplification) and FISH (Fluorescent in situ Hybridization). Results The ring chromosomes of patients I to XIV were determined to be, respectively: r(3)(p26.1q29), r(4)(p16.3q35.2), r(10)(p15.3q26.2), r(10)(p15.3q26.13), r(13)(p13q31.1), r(13)(p13q34), r(14)(p13q32.33), r(15)(p13q26.2), r(18)(p11.32q22.2), r(18)(p11.32q21.33), r(18)(p11.21q23), r(22)(p13q13.33), r(22)(p13q13.2), and r(22)(p13q13.2). These rings were found to have been formed by different mechanisms, such as: breaks in both chromosome arms followed by end-to-end reunion (patients IV, VIII, IX, XI, XIII and XIV); a break in one chromosome arm followed by fusion with the subtelomeric region of the other (patients I and II); a break in one chromosome arm followed by fusion with the opposite telomeric region (patients III and X); fusion of two subtelomeric regions (patient VII); and telomere-telomere fusion (patient XII). Thus, the r(14) and one r(22) can be considered complete rings, since there was no loss of relevant genetic material. Two patients (V and VI) with r(13) showed duplication along with terminal deletion of 13q, one of them proved to be inverted, a mechanism known as inv-dup-del. Ring instability was detected by ring loss and secondary aberrations in all but three patients, who presented stable ring chromosomes (II, XIII and XIV). Conclusions We concluded that the clinical phenotype of patients with ring chromosomes may be related with different factors, including gene haploinsufficiency, gene duplications and ring instability. Epigenetic factors due to the circular architecture of ring chromosomes must also be considered, since even complete ring chromosomes can result in phenotypic alterations, as observed in our patients with complete r(14) and r(22).

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.