Pleural solitary fibrous tumours (pSFTs) are uncommon soft tissue tumours of the pleura. that may recur and contribute to the patients’ demise. We analyzed a group of benign and malignant pSFTs for copy number alterations and for common mutations in oncogenes and tumour-suppressor genes. Malignant SFTs demonstrated more copy number alterations, especially 8q (c-myc) gain, 10q (include PTEN) loss, and 13q (Rb1) loss. Mutations were rare in this limited study.

Separate domestication events and strong selective pressures have created diverse phenotypes among existing cattle populations; however, the genetic determinants underlying most phenotypes are currently unknown. Bos taurus taurus (Bos taurus) and Bos taurus indicus (Bos indicus) cattle are subspecies of domesticated cattle that are characterized by unique morphological and metabolic traits. Because of their divergence, they are ideal model systems to understand the genetic basis of phenotypic variation. Here, we developed DNA and structural variant maps of cattle genomes representing the Bos taurus and Bos indicus breeds. Using this data, we identified genes under selection and biological processes enriched with functional coding variants between the two subspecies. Furthermore, we examined genetic variation at functional non-coding regions, which were identified through epigenetic profiling of indicative histone- and DNA-methylation modifications. Copy number variants, which were frequently not imputed by flanking or tagged SNPs, represented the largest source of genetic divergence between the subspecies, with almost half of the variants present at coding regions. We identified a number of divergent genes and biological processes between Bos taurus and Bos indicus cattle; however, the extent of functional coding variation was relatively small compared to that of functional non-coding variation. Collectively, our findings suggest that copy number and functional non-coding variants may play an important role in regulating phenotypic variation among cattle breeds and subspecies.

No description available.

No description available.

We developed a pipeline to identify the copy number polymorphisms (CNPs) in the Northern Swedish population using whole genome sequencing (WGS) data. Two different methodologies were applied to discover CNPs in more than 1,000 individuals. We also studied the association between the identified CNPs with the expression level of 438 plasma proteins collected in the same population. The identified CNPs were summarized and filtered as a population copy number matrix for 1,021 individuals in 243,987 non-overlapping CNP loci. For the 872 individuals with both WGS and plasma protein biomarkers data, we conducted linear regression analyses with age and sex as covariance. From the analyses, we detected 382 CNP loci, clustered in 30 collapsed copy number variable regions (CNVRs) that were significantly associated with the levels of 17 plasma protein biomarkers (p < 4.68×10-10).

Mestrado em Ciências Forenses / Master Degree Course in Forensic Sciences / A possibilidade de analisar amostras com quantidades exíguas de material genético (amostras Low Copy Number ou LCN), em que estão presentes apenas algumas células, tem alterado a forma de encarar a cena do crime. Alguns vestígios que até agora não eram considerados como susceptíveis de proporcionarem resultados, podem actualmente ser analisados com sucesso. As impressões digitais são um bom exemplo. Estes vestígios apresentam um baixo número de células, permitindo apenas recuperar quantidades de DNA inferiores a 100 pg. Assim, para a análise do DNA nuclear, é necessário implementar sistemas muito sensíveis que consistem, habitualmente, no aumento do número de ciclos da PCR. Contudo, alguns artefactos são produzidos, tornando difícil a interpretação dos electroforectogramas. Neste trabalho pretendeu-se comparar a aplicação do estudo de STR autossómicos, Y-STR e miniSTR na análise genética de impressões digitais, partindo do conceito do aumento do número de ciclos como estratégia para se obter maior sensibilidade. Procedeu-se também à amplificação total do genoma e nested-PCR, como métodos alternativos ao aumento do número de ciclos. Adicionalemente, neste estudo tentou-se perceber a influência dos principais métodos reveladores de impressões digitais (cianoacrilato, pó magnético e pó branco) na análise do DNA. Os resultados mostram que o aumento do número de ciclos é a melhor opção como método para aumentar a sensibilidade. Constata-se também que o DNA extraído de impressões digitais encontra-se parcialmente degradado, obtendo-se diferenças significativas entre loci com fragmentos de amplificação menores e maiores do que 200 pb. Dos diferentes marcadores caracterizados verifica-se que, em termos de percentagem de alelos detectados, os miniSTR proporcionam os melhores resultados. Por outro lado, os Y-STR parecem altamente sensíveis à degradação ou presença de inibidores, pelo que são menos robustos para este tipo de análises. Verifica-se também que os perfis LCN são drasticamente afectados por artefactos, principalmente os derivados de variação estocástica, como o allele dropout e o desequilíbrio heterozigótico. A determinação de perfis de consenso permite reduzir alguns destes artefactos. Dos métodos de revelação estudados, o cianoacrilato é o que apresenta menor influência na análise e, pelo contrário, o pó branco provoca os resultados mais negativos. / The possibility to perform low copy number DNA typing, when just a few cells are available, as changed the way how crime scene investigations is faced. Nowadays it is possible to successfully type some evidence that couldn t be considered until now. Fingerprints are a good example of those. Since that just a few cells are present in this evidence (enabling recovery of low quantities of DNA, fewer than 100pg) just very sensitive systems can detect nuclear DNA. The most used method is definitely increasing the number of PCR cycles. However, increased occurrence of stutters and artifacts that reduced the quality of the DNA profile is normally observed. The present work aimed to compare the application of autosomic STR, Y-STR and miniSTR markers, based on the concept of increased number of PCR cycles as a strategy to achieve more sensitivity. Some other methods, such as whole genome amplification and nested-PCR, were also evaluated as an alternative way to reach the desired sensitivity. Another goal was to determine the influence of several reagents for developing latent fingerprints (cyanoacrylate fuming, magnetic powder and white powder) in DNA typing. The results shows that increasing the number of PCR cycles still is the best way to attain the required sensitivity. Moreover we could realize that DNA was partially degraded, once there were observed significant differences between loci larger and smaller than 200bp. Among all markers miniSTR showed to perform the best results in terms of detected alleles percentage. On the other hand, Y-STR seemed to be highly affected in the presence of degraded DNA and PCR inhibitors, which makes them less robust for these analyses. LCN profiles are significantly affected by artifacts, like allele dropout and heterozygous imbalance, derived from stochastic fluctuation. Reporting consensus profiles reduces artifact inherent errors. Finally, cyanoacrylate proved to have a minimum negative effect on DNA profiling, while white powder was the worst reagent.

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

When offenders commit crime there is the potential that they may leave behind trace amounts of their DNA, even when there has been no apparent body fluid spill. During the examination of crime scenes, scene investigators try to identify areas that may be sampled to locate these traces. Specialist techniques are then required within the laboratory to enable such small amounts to be analysed to obtain a profile. These techniques are referred to as Low Template DNA analysis (LTDNA), of which Low Copy Number DNA (LCN DNA) is one instance. In 2008, following the Omagh Bombing trial, and comments made by Judge Weir, the UK Forensic Regulator commissioned a review of the science of LTDNA analysis. The subsequent report made specific mention of the fact that there was no available information on the success rate of the use of such DNA techniques and that there seemed to be confusion over what constituted a success. The report went on to state that there was no information on where such trace amounts of DNA were likely to be found, or what factors could influence the likelihood of obtaining a trace DNA profile (Caddy, 2008). This research considered the outcomes of LCN DNA analysis from 3,552 samples to try to establish where trace amounts of DNA could be found, whether some areas sampled were more successful in generating profiles than others, and the likelihood of the profiles obtained being of use to a criminal investigation. Analysis of results identified areas that were more successful in generating profiles of use to an investigation and highlighted significant differences in results across a variety of items from which samples were taken. DNA samples taken from items associated with communication such as mobile phones were much more likely to produce a profile useful to a criminal investigation than those taken from fixed surfaces within premises. The results obtained showed that obtaining a DNA profile did not necessarily correlate with the profile being of use to a criminal investigation. This was due to the fact that a large number of these profiles were anticipated eliminations from legitimate sources. Items that produced high numbers of profiles but were anticipated eliminations, and therefore of no value to an investigation, came from items associated with skin samples and clothing. The research went further to identify key factors that affected the profiling rates. Factors that had a positive influence on the ability to obtain a profile included: any area that had been in close proximity to saliva (direct contact was not required); samples that had been recovered from the inside of premises or vehicles and therefore protected from the elements; those that were dry; items that were of a porous nature; and those that had a rough texture. No differences were found between the actual surface materials (plastic, glass, wood, metal), as all showed a propensity to generate profiles. Other factors that were considered but proved to have no effect on the profiling rates included seasonal differences and whether the area targeted for sampling was clearly defined. Items that had had high contact with a victim, were recovered from outside or had been wet, all proved to be less useful to an nvestigation. A further finding of the research was that swabs that had been recovered and stored frozen appeared to deteriorate in their ability to profile. This was particularly notable if they were submitted later than 5 months after recovery. Items stored in dry conditions did not deteriorate in this way. Overall the research can be used to provide investigators with the knowledge of what areas of crime scenes are most likely to yield trace DNA material, the key factors that can affect the likelihood of obtaining a profile, and those areas that are more likely to produce profiles useful to criminal investigations.

Improved survival for women receiving chemotherapy for breast cancer (BC) has been accompanied by the development/persistence of psychoneurological symptoms (PNS) that compromise their quality of life. The biological basis for these PNS is unknown, but could reflect the acquisition of soma-wide chromosomal/epigenetic alterations. An important first step in testing this hypothesis is to determine if somatic genetic/epigenetic changes arise and persist following treatment. To answer this question we longitudinally studied 71 women (ages 23-71) with early-stage BC and collected measures before chemotherapy (baseline), and 4 weeks (mid-chemo); six months (during radiation therapy for a subset of women); and one year following the initiation of chemotherapy. Acquired lymphocyte chromosomal instability (scored by micronuclei frequencies [MNF]) showed a significant increase in post-treatment compared to baseline time-points (p<0.0001), with these increases persisting for at least one year following chemotherapy. Significant predictive associations were observed between MNF and tumor characteristics [luminal B (lower MNF; p=0.0182); triple negative (higher MNF; p=0.0446)], radiotherapy (higher MNF; p=0.0004), the type of chemotherapy received (p=0.0463), race (Caucasians > African Americans; p=0.0037), perceived stress levels (positive-association; p=0.0123), and cognitive flexibility domain measures (positive-association; p=0.0238). Genome-wide acquired methylation changes were also measured in peripheral blood cells, with 1265 sites showing significant differential methylation following chemotherapy. These sites were localized to open sea, shores, shelves, and CpG island sequences and included sites within genes involved in cell cycle, DNA repair, transcription regulation, signal transduction pathways, neuronal regeneration, and immunity. To determine if the genetic/epigenetic alterations acquired in peripheral blood cells correlated with those in tumor cells, BC tumors from 10 participants were analyzed using a genome-wide copy number/targeted mutations (CN/M) microarray. While no clear blood-tumor cell correlations were detected, genome-wide CN/M evaluations showed promise for stratifying tumors. Lastly, in an unrelated project studying a rare case of fetuses in fetu, methylation changes acquired in embryogenesis were shown to be influenced by both environmental and genetic cues. In summary, acquired chromosomal/epigenetic alterations do arise following chemotherapy (and in embryogenesis). Further delineation of these acquired changes could increase our understanding of the biological basis for cancer-related side-effects and help to identify “at risk” individuals.

Plasmids are small circular DNA molecules within bacterial cells that are separated from the bacterial chromosome and replicate independently. Also, they play a crucial role in the dissemination of antibiotic resistance genes among bacteria through horizontal gene transfer. They can be present in many copies within host cell, which is known as plasmid copy number. Plasmids can regulate their own copy number by different mechanisms. Additionally, the selective pressure can also play a pivotal role in determining plasmid copy number. The presence of antibiotics in the surrounding environment can drive variations of plasmid copy number. In this study, we examined plasmid copy number variations of multidrug resistance plasmids in presence of antibiotics by using EvaGreen® - based multiplexed digital droplet PCR. We could observe that cultures of Klebsiella pneumoniae and Escherichia coli harboring multidrug resistance plasmids grown in presence of sub-MIC concentrations of the antibiotics did not show high variations in plasmid copy numbers. On the other hand, mutants of K. pneumoniae selected for increased antibiotic resistance showed high increases in copy number of a multidrug-resistance plasmid.