Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families

Genetic screening of women from multiple-case breast cancer families and other research-based endeavors have identified an extensive collection of germline variations of BRCA1 and BRCA2 that can be classified as deleterious and have clinical relevance. For some variants, such as those in the conserved intronic splice site regions which are highly likely to alter splicing, it is not possible to classify them based on the identified DNA sequence variation alone. We studied 11 multiple-case breast cancer families carrying seven distinct splice site region genetic alterations in BRCA1 or BRCA2 (BRCA1, c.IVS6-2delA, c.IVS9-2A>C, c.IVS4-1G>T, c.IVS20+1G>A and BRCA2, c.IVS17-1G>C, c.IVS20+1G>A, c.IVS7-1G>A) and applied SpliceSiteFinder to predict possible changes in efficiency of splice donor and acceptor sites, characterized the transcripts, and estimated the average age-specific cumulative risk (penetrance) using a modified segregation analysis. SpliceSiteFinder predicted and we identified transcipts that illustrated that all variants caused exon skipping, and all but two led to frameshifts. The risks of breast cancer to age 70 yrs, averaged over all variants, over BRCA1 variants alone, and over BRCA2 variants alone, were 73% (95% confidence interval 47-93), 64% (95%CI 28-96) and 79% (95%CI 48-98) respectively (all P

Characterization of two polymorphisms in the leukotriene C4 synthase gene in an Australian population of subjects with mild, moderate, and severe asthma

Background: The cysteinyl-leukotrienes (cys-LTs) are proinflammatory mediators that are important in the pathophysiology of asthma. LTC4 synthase is a key enzyme in the cys-LT biosynthetic pathway, and studies in small populations have suggested that a promoter polymorphism (A(-444)C) in the gene might be associated with asthma severity and aspirin intolerance. Objective: We sought to screen the LTC4 synthase gene for polymorphisms and to determine whether there is an association between these polymorphisms and asthma severity or aspirin sensitivity in a large, well-phenotyped population and to determine whether this polymorphism is functionally relevant. Methods: The coding regions of the LTC4 synthase gene were screened for polymorphisms and the A(-444)C polymorphism was analyzed in a large Australian white adult population of mild (n = 282), moderate (n = 236), and severe asthmatic subjects (n = 86) and nonasthmatic subjects (n = 458), as well as in aspirin-intolerant asthmatic subjects (n = 67). The functional activity of the promoter polymorphism was investigated by transient transfection of HL-60 cells with a promoter construct. Results: A new polymorphism was identified in intron 1 of the gene (IVS1-10c>a) but was not associated with asthma. Association studies showed that the A(-444)C polymorphism was weakly associated with asthma per se, but there was no association between the C-444 allele and chronic asthma severity or aspirin intolerance. A meta-analysis of all the genetic studies conducted to date found significant between-study heterogeneity in C-444 allele frequencies within different clinical subgroups. In vitro functional studies showed no significant differences in transcription efficiency between constructs containing the A(-444) allele or the C-444 allele. Conclusions: Our data confirm that, independent of transcriptional activity, the C-444 allele in the LTC4 synthase gene is weakly associated with the asthma phenotype, but it is not related to disease severity or aspirin intolerance.

Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 10(4)-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in alpha/beta interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73%) and amino acid sequence (83 %) identity between ALFV and IMVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5'-CUOH-3' found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to IMVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.

Molecular characterization of Thelastomatoidea (Nematoda : Oxyurida) from cockroaches in Australia

A molecular approach was used to genetically characterize 5 species (Aoruroides queenslandensis. Blattophila sphaerolaima, Cordonicola gibsoni, Desmicola ornato and Leidynemella fusiformis) belonging to the superfamily. Thelastomatoidea fi (Nematoda: Oxyurida), a group of pinworms that parasitizes terrestrial arthropods. The D3 domain of the large subunit Of nuclear ribosomal RNA (LSU) was sequenced for individual specimens, and the analysis of the sequence data allowed the genetic relationships of the 5 species to be studied dagger. The sequence variation in the D3 domain within individual species (0-1-8%) was significantly less than the differences among species (4(.)3-12(.)4%). Phylogenetic analyses, Using maximum parsimony, maximum likelihood, and neighbour-joining, tree-building methods, established relationships among the 5 species of Thelastomatoidea and Oxyuris equi (a species of the order Oxyurida). The molecular approach employed provides the prospect for developing DNA tools for the specific identification of the Thelastomatoidea, irrespective of developmental stage and sex, as a basis for systematic, ecological and/or population genetic investigations of members within this superfamily.

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed. (c) 2006 Published by Elsevier Ltd on behalf of The British Mycological Society.

Isolation and characterization of microsatellite loci from Macadamia

Thirty-three microsatellite loci were isolated for the Australian rainforest tree Macadamia integrifolia. Genotyping across a test panel of 43 commercial cultivars generated an average polymorphic information content of 0.480. Five loci showed no polymorphism across cultivars. Significant linkage disequilibrium was detected in 10 pairwise comparisons, including two pairs of loci identified from the same clone sequence. The 33 microsatellite loci represent a significant tool for genome mapping and population genetic studies.

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

Isolation, characterization and discovery of new alleles of sHsp genes in durum wheat (Triticum durum Desf.)

Gli organismi vegetali mostrano una notevole capacità di adattamento alle condizioni di stress e lo studio delle componenti molecolari alla base dell'adattamento in colture cerealicole di interesse alimentare, come il frumento, è di particolare interesse per lo studio di varietà che consentano una buona produzione con basso input anche in condizioni ambientali non ottimali. L'esposizione delle colture cerealicole a stress termico durante determinate fasi del ciclo vitale influisce negativamente sulla resa e sulla qualità, a questo fine è necessario chiarire le basi genetiche e molecolari della termotolleranza per identificare geni e alleli vantaggiosi da impiegare in programmi di incrocio volti al miglioramento genetico. Numerosi studi dimostrano il coinvolgimento delle sHSP a localizzazione cloroplastica (in frumento sHSP26) nel meccanismo di acquisizione della termotolleranza e la loro interazione con diverse componenti del fotosistema II (PSII) che determinerebbe un’azione protettiva in condizioni di stress termico e altri tipi di stress. Lo scopo del progetto è quello di caratterizzare in frumento duro nuove varianti alleliche correlate alla tolleranza a stress termico mediate l'utilizzo del TILLING (Target Induced Local Lesion In Genome), un approccio di genetica inversa che prevede la mutagenesi e l'identificazione delle mutazioni indotte in siti di interesse. Durante la tesi sono state isolate e caratterizzate 3 sequenze geniche complete per smallHsp26 denominate TdHsp26-A1; TdHsp26-A2; TdHsp26-B1 e un putativo pseudogene denominato TdHsp26-A3. I geni isolati sono stati usati come target in analisi di TILLING in due popolazioni di frumento duro mutagenizzate con EMS (EtilMetanoSulfonato). Nel nostro studio sono stati impiegati due differenti approcci di TILLING: un approccio di TILLING classico mediante screening con High Resolution Melting (HRM) e un approccio innovativo che sfrutta un database di TILLING recentemente sviluppato. La popolazione di mutanti cv. Kronos è stata analizzata per la presenza di mutazioni in tutti e tre i geni individuati mediante ricerca online nel database di TILLING, il quale sfrutta la tecnica dell’exome capture sulla popolazione di TILLING seguito da sequenziamento ad alta processività. Attraverso questa tecnica sono state individuate, nella popolazione mutagenizzata di frumento duro cv. Kronos, 36 linee recanti mutazioni missenso. Contemporaneamente lo screening con HRM, effettuato su 960 genotipi della libreria di TILLING di frumento duro cv. Cham1 ha consentito di individuare mutazioni in una regione di 211bp di interesse funzionale del gene TdHsp26-B1, tra le quali 3 linee mutanti recanti mutazioni missenso in omozigosi. Alcune mutazioni missenso individuate sui due geni TdHsp26-A1 e TdHsp26-B1 sono state confermate in vivo nelle piante delle rispettive linee mutanti generando marcatori codominanti KASP (Kompetitive Allele Specific PCR) con cui è stato possibile verificare anche il grado di zigosità di tali mutazioni. Al fine di ridurre il numero di mutazioni non desiderate nelle linee risultate più interessanti, è stato eseguito il re-incrocio dei mutanti con i relativi parentali wild type ed inoltre sono stati generati alcuni doppi mutanti che consentiranno di comprendere meglio i meccanismi molecolari presieduti da questa classe genica. Gli individui F1 degli incroci sono stati poi genotipizzati con i medesimi marcatori KASP specifici per la mutazione di interesse per verificare la buona riuscita dell’incrocio. Questo approccio ha permesso di individuare ed implementare risorse genetiche utili ad intraprendere studi funzionali relativi al ruolo di smallHSP plastidiche implicate nella acquisizione di termotolleranza in frumento duro e di generare marcatori potenzialmente utili in futuri programmi di breeding.

Noisy fitness evaluation in genetic algorithms and the dynamics of learning

A theoretical model is presented which describes selection in a genetic algorithm (GA) under a stochastic fitness measure and correctly accounts for finite population effects. Although this model describes a number of selection schemes, we only consider Boltzmann selection in detail here as results for this form of selection are particularly transparent when fitness is corrupted by additive Gaussian noise. Finite population effects are shown to be of fundamental importance in this case, as the noise has no effect in the infinite population limit. In the limit of weak selection we show how the effects of any Gaussian noise can be removed by increasing the population size appropriately. The theory is tested on two closely related problems: the one-max problem corrupted by Gaussian noise and generalization in a perceptron with binary weights. The averaged dynamics can be accurately modelled for both problems using a formalism which describes the dynamics of the GA using methods from statistical mechanics. The second problem is a simple example of a learning problem and by considering this problem we show how the accurate characterization of noise in the fitness evaluation may be relevant in machine learning. The training error (negative fitness) is the number of misclassified training examples in a batch and can be considered as a noisy version of the generalization error if an independent batch is used for each evaluation. The noise is due to the finite batch size and in the limit of large problem size and weak selection we show how the effect of this noise can be removed by increasing the population size. This allows the optimal batch size to be determined, which minimizes computation time as well as the total number of training examples required.

Survival in the pre-senile dementia frontotemporal lobar degeneration with TDP-43 proteinopathy:effects of genetic, demographic and neuropathological variables

Factors associated with survival were studied in 84 neuropathologically documented cases of the pre-senile dementia frontotemporal dementia lobar degeneration (FTLD) with transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) proteinopathy (FTLD-TDP). Kaplan-Meier survival analysis estimated mean survival as 7.9 years (range: 1-19 years, SD = 4.64). Familial and sporadic cases exhibited similar survival, including progranulin (GRN) gene mutation cases. No significant differences in survival were associated with sex, disease onset, Braak disease stage, or disease subtype, but higher survival was associated with lower post-mortem brain weight. Survival was significantly reduced in cases with associated motor neuron disease (FTLD-MND) but increased with Alzheimer's disease (AD) or hippocampal sclerosis (HS) co-morbidity. Cox regression analysis suggested that reduced survival was associated with increased densities of neuronal cytoplasmic inclusions (NCI) while increased survival was associated with greater densities of enlarged neurons (EN) in the frontal and temporal lobes. The data suggest that: (1) survival in FTLD-TDP is more prolonged than typical in pre-senile dementia but shorter than some clinical subtypes such as the semantic variant of primary progressive aphasia (svPPA), (2) MND co-morbidity predicts poor survival, and (3) NCI may develop early and EN later in the disease. The data have implications for both neuropathological characterization and subtyping of FTLD-TDP.