A DNA sequence-based phylogenetic tree (Dillon et al., 2004) places the species of the genus Sorghum into two sister lineages, one with x = 5 and the other with x = 10 as a basic chromosome number. It has not been resolved whether or not these lineages are monophyletic or polyphyletic. A repetitive sequence, CEN38, found only in Sorghum and sugarcane, was used to assess evolutionary relationships among Sorghum species. The objectives of this research were to determine the taxonomic distribution of CEN38, its chromosomal position(s), and its organization in DNA. CEN38 was detected by filter hybridization to be present in the DNA of 16 of 21 Sorghum species analyzed, ranging from 15 to ~21,000 copies. It was detected by fluorescence in situ hybridization (FISH) only in chromosomes of species of the section Eu-sorghum, where it had a pericentromeric distribution. The low copy number and/or chromosomal distribution of CEN38 in other Sorghum species apparently does not allow for its detection by FISH. Analysis of restriction enzyme digested DNA with homology to CEN38 and of fragments amplified by PCR using primers selected to amplify S. bicolor CEN38 sequences showed that S. laxiflorum and S. macrospermum have tandemly arranged CEN38 sequences as is found in S. bicolor. This supports the close evolutionary affinity of the species in the x = 10 lineage. In the x = 5 lineage, DNA of 11 of 16 species analyzed hybridized with CEN38 by filter hybridization. In S. versicolor, large DNA fragments (4.36 kb to 23 kb) generated by digestion with restriction enzymes hybridized to CEN38. Since a ladder of smaller fragments was not detected, CEN38 may have been inserted into a transposable element in this species and dispersed throughout the genome. Among species of the x = 5 lineage, PCR using primers for S. bicolor CEN38 amplified only DNA fragments from S. timorense and these formed a ladder based on a ~125 bp repeat. Since hybridization of the CEN38 sequence to DNA of S. timorense was not detected by filter hybridizations, these sequences apparently are not similar to CEN38. Cloning and sequencing of DNA from species of the x = 5 lineage that hybridizes to CEN38 are needed to determine whether or not they are in the CEN38 family. A monophyletic or polyphyletic origin of the x = 5 and x = 10 lineages was not resolved.

Genome wide studies have uncovered the existence of large-scale copy number variation (CNV) in the human genome. The human genome of different individuals was initially estimated to be 99.9% similar, but population studies on CNV have revealed that it is 12-16% copy number variable. Abnormal genomic CNVs are frequently found in subtelomeres of patients with mental retardation (MR) and other neurological disorders. Rearrangements of chromosome subtelomeric regions represent a high proportion of cytogenetic abnormalities and account for approximately 30% of pathogenic CNVs. Although DNA double strand breaks (DSBs) are implicated as a major factor in chromosomal rearrangements, the causes of chromosome breakage in subtelomeric regions have not been elucidated. But due to the presence of repetitive sequences in subtelomeres, we hypothesized that chromosomal rearrangements in these regions are not stochastic but driven by specific sequence motifs. In a collaborative effort with Dr. Rudd (Department of human genetics at Emory University), we characterized subtelomeric breakpoints on different chromosome ends in search of common motifs that cause double-strand breaks. Using a yeast-based gross chromosomal rearrangement (GCR) system, we have identified a subtelomeric breakage motif from chromosome 2 (2q SBM) with a GCR rate that is 340 fold higher than background levels. To determine if the fragility of 2q SBM was driven by the formation of secondary structures, the helicase activities of Sgs1 and Pif1 were disrupted. These helicases have been shown to destabilize DNA secondary structures such as G-quadruplex structures. Disruption of these helicases augmented chromosomal rearrangements induced by 2q SBM, indicating that these helicases are required for maintenance of this sequence. We also donwregulated replication fork components to determine if 2q SBM was imposing any problems to the replication fork machinery. Downregulation of replication fork components increased chromosomal rearrangements, indicating that intact replication fork was a critical determinant of 2q SBM fragility. Using a yeast-based functional assay, these experiments have linked human subtelomeric repetitive sequences to chromosomal breakage that could give rise to human CNV in subtelomeric regions.

Mitochondrial diseases involving dysfunction of the respiratory chain are the most common inborn errors of metabolism. Mitochondria are found in all cell types besides red blood cells; consequently, patients can present with any symptom in any organ at any age. These diseases are genetically heterogeneous, and exhibit maternal, autosomal dominant, autosomal recessive and X-linked modes of inheritance. Historically, clinical genetic evaluation of mitochondrial disease has been limited to sequencing of the mitochondrial DNA (mtDNA) or several candidate genes. As human genome sequencing transformed from a research grade effort costing $250,000 to a clinical test orderable by doctors for under $10,000, it has become practical for researchers to sequence individual patients. This thesis describes our experiences in applying "MitoExome" sequencing of the mtDNA and exons of >1000 nuclear genes encoding mitochondrial proteins in ~200 patients with suspected mitochondrial disease. In 42 infants, we found that 55% harbored pathogenic mtDNA variants or compound heterozygous mutations in candidate genes. The pathogenicity of two nuclear genes not previously linked to disease, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. In an additional two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency, we identified compound heterozygous mutations in MTFMT. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-\(tRNA^{Met}\) levels and an abnormal formylation profile of mitochondrially translated \(COX_1\). These results demonstrate that MTFMT is critical for human mitochondrial translation. Lastly, to facilitate evaluation of copy number variants (CNVs), we developed a web-interface that integrates CNV calling with genetic and phenotypic information. Additional diagnoses are suggested and in a male with ataxia, neuropathy, azoospermia, and hearing loss we found a deletion compounded with a missense variant in D-bifunctional protein, \(HSD_{17}B_4\), a peroxisomal enzyme that catalyzes beta-oxidation of very long chain fatty acids. Retrospective review of metabolic testing from this patient revealed alterations of long- and very-long chain fatty acid metabolism consistent with a peroxisomal disorder. This work expands the molecular basis of mitochondrial disease and has implications for clinical genomics.

Im Rahmen dieser Arbeit wurde Organisation, Expression und Funktion des humanen Peroxisomal-Testis-Spezifisch-1(PXT1)-Gens untersucht. Die mRNA des humanen PXT1-Gens enthält nicht wie bisher bekannt zwei Exons, sondern fünf Exons. Die Expression von drei putativen Exons stromaufwärts konnte in dieser Arbeit bestätigt werden. Die Ergebnisse qualitativer und quantitativer Real Time-PCR zeigen, dass sich das Exon 1 aus drei unterschiedlich gespleißten Einheiten (Exons 1a, 1b und 1c) zusammensetzt. Das humane PXT1-Gen unterliegt dem alternativen Spleißen, wovon die Exons 1b, 1c, 2 und 4 betroffen sind, was Sequenzanalysen zeigen. Sechs Transkripte konnten insgesamt identifiziert werden. Die zusätzlichen Exons haben Auswirkungen auf die Proteinstruktur aufgrund der Verlängerung des ORF, kodierend für einst 51 Aminosäuren, auf 134. Im längeren Protein wird die BH3 interacting domain (BID) nachgewiesen, von der eine proapoptotische Funktion bekannt ist. Aufgrund des alternativ gespleißten Exon 4 und der daraus resultierenden Leserasterverschiebung existiert ein verkürztes Protein, in dessen mRNA sich ein vorzeitiges Stopkodon befindet. Die proapoptotische Domäne ist nicht mehr nachweisbar. In silico-Analysen zeigen, dass die Sequenzen der Exons 1a und 1b von PXT1 sich mit dem KCTD20-Gen überlappen, das für einen Kaliumkanal kodiert.  Im Unterschied zum murinen, testisspezifischen Pxt1-Gen, ist das humane Homolog trotz Prädominanz im Testis auch schwächer in anderen Geweben nachweisbar.  Zur weiteren Klärung der proapoptotischen Funktion von Pxt1 in Keimzellen wurde am Mausmodell (Pxt1-Knockout-Maus) die Anzahl an DNA-Strangbrüchen untersucht. Im Vergleich zu den Kontrolltieren (C57BL/6J) zeigt die Pxt1-Knockout-Maus eine signifikant erhöhte Anzahl an Spermien mit DNA-Strangbrüchen. Dieses Ergebnis bestätigt die Annahme, dass das PXT1/Pxt1-Gen eine Art Entsorgungsfunktion für beschädigte Spermien ausübt. Im zeitlichen Verlauf zeigte sich aber, dass die Spermien der Knockout-Tiere nicht sensibler als die Wildtyp-Tiere auf DNaseI reagieren.  Als mögliches Kandidatengen für Mutationsanalysen bei Männern mit Fertilitätsstörungen wurden 55 Patienten mit Fertilitätsstörungen (Azoo- oder Oligozoospermie) auf Punktmutationen im PXT1 untersucht. Eine Mutation konnte nicht identifiziert werden. Des Weiteren wurde die DNA der Patienten auf Copy Number Variations analysiert. Sowohl heterozygote als auch homozygote Duplikationen konnten im Exon1, bestätigt mithilfe der arraybasierten Comparativen Genomischen Hybridisierung (aCGH), vereinzelt auch in Exon2 und Exon3 nachgewiesen werden. Zusätzlich konnte bei einem Patienten eine Deletion nachgewiesen werden. Die bestätigte Duplikation im Exon 1 besitzt aber keinen Krankheitswert, da sie in einem Kontrollkollektiv eine Prävalenz von 41% in heterozygoter und 10% in homozygoter Form besitzt.

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

Post-zygotic genetic variation has previously been shown in healthy individuals and linked to various disorders. The definition of post-zygotic or somatic variation is the existence of genetically distinct populations of cells in a subject derived from a single zygote. Structural changes in the human genome are a major type of inter-individual genetic variation and copy number variation (CNV), involving changes in the copy number of genes, are one of the best studied category of structural genetic changes. In paper I we reported a pair of healthy female monozygotic (MZ) twins discordant for aneuploidy of chromosomes X and Y, contributing to the delineation of the frequency of somatic variation in MZ twins. It also illustrates the plasticity of the genome for tolerating large aberrations in healthy subjects. In paper II we showed age-related accumulation of copy number variation in the nuclear genomes in vivo for both megabase- and kilobase-range variants. Using age-stratified MZ twins and single-born subjects, we detected megabase-range aberrations in 3.4% of people ≥60 years old but not in individuals younger than 55 years. Moreover, the longitudinal analysis of subjects with aberrations suggests that the aberrant cell clones are not immortalized and disappear from circulation. We also showed that sorted blood cells display different genomic profiles.  The detected recurrent rearrangements are candidates for common age-related defects in blood cells. This work might help to describe the cause of an age-related decline in the number of cell clones in the blood, which is one of the hallmarks of immunosenescence. In paper III we described a variable number tandem repeat (VNTR) ~4 kb upstream of the IFNAR1 gene, which was somatically variable.  We detected 14 alleles displaying inter- and intra-individual variation. Further analyses indicated strong clustering of transcription factor binding sites within this region, suggesting an enhancer. This putative VNTR-based enhancer might influence the transcriptional regulation of neighboring cytokine receptor genes and the pathways they are involved in. These three studies stress the importance of research on post-zygotic variation in genetics. Furthermore, they emphasize that biobanks should consider sampling of multiple tissues to better address this issue in the genetic studies.

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

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

Prostate cancer is a clinically heterogeneous disease, with manifestations ranging from a rapid and often fatal progression, to indolent disease. Unfortunately, current clinicopathological criteria cannot differentiate men whose tumours require immediate and aggressive therapy from those in which active surveillance may be more appropriate. Both PTEN deletion and ETS gene fusions are biomarkers with potential to aid in prostate cancer clinical management. In this thesis, I postulate that PTEN and fusion gene rearrangements may be associated with specific genomic changes, and might also have general impact on the genomic landscape of prostate cancer. A meta-analysis of somatic copy number alterations (CNAs) examined 662 unique prostate cancer patient samples consisting of 546 primary and 116 advanced tumours derived from eleven publications. Normalization, segmentation and identification of corresponding CNAs for meta-analysis were achieved using established commercial software. The CNA distribution in primary disease was characterized by losses at 2q, 3p, 5q, 6q, 8p, 12p, 13q, 16q, 17p, 18q and 10q (PTEN), and acquisition of 21q deletions associated with the TMPRSS2:ERG fusion rearrangement. Unsupervised analysis identified five genomic subgroups. Parallel analysis of advanced and primary tumours indicated that PTEN genomic deletions and the gene fusion were enriched in advanced disease. A supervised analysis of PTEN deletions and gene fusions demonstrated that PTEN deletion was sufficient to impose higher levels of CNA. Moreover, the overall percentage of the genome altered was significantly higher when PTEN was deleted, suggesting that this important genomic subgroup was likely characterized by intrinsic chromosomal instability. Candidate genes in each of the recurrent CNA regions characteristic of each subgroup showed that signalling networks associated with cancer progression and genome stability were likely to be perturbed at the highest level in the PTEN deleted genomic subgroup. Therefore classification of primary prostate cancer according to PTEN deletions, but not the gene fusion, was associated with greatly increased levels of CNA. Collectively, the impact of PTEN loss resulted in a significantly greater frequency and extent of alteration, and heightened genomic instability with concomitant pathway disruptions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2014-04-29 14:20:06.02

Understanding the mechanism of recombination has important implications for genome evolution and genomic variability. The work presented in this thesis studies the properties of recombination by investigating the effects it has on genome evolution in humans and dogs. Using alignments of human genes with chimpanzee and macaque orthologues we studied substitution patterns along the human lineage and scanned for evidence of positive selection. The properties mirror the situation in human non-coding sequences with the fixation bias ‘GC-biased gene conversion’ (gBGC) as a driving force in the most rapidly evolving regions. By assigning candidate genes to distinct classes of evolutionary forces we quantified the extent of those genes affected by gBGC to 20%. This suggests that human-specific characters can be prompted by the fixation bias of gBGC, which can be mistaken for selection. The gene PRDM9 controls recombination in most mammals, but is lacking in dogs. Using whole-genome alignments of dog with related species we examined the effects of PRDM9 inactivation. Additionally, we analyzed genomic variation in the genomes of several dog breeds. We identified that non-allelic homologous recombination (NAHR) via sequence identity, often GC-rich, creates structural variants of genomic regions. We show that these regions, which are also found in dog recombination hotspots, are a subset of unmethylated CpG-islands (CGIs). We inferred that CGIs have experienced a drastic increase in biased substitution rates, concurrent with a shift of recombination to target these regions. This enables recurrent episodes of gBGC to shape their distribution. The work presented in this thesis demonstrates the importance of meiotic recombination on patterns of molecular evolution and genomic variability in humans and dogs. Bioinformatic analyses identified mechanisms that regulate genome composition. gBGC is presented as an alternative to positive selection and is revealed as a major factor affecting allele configuration and the emergence of accelerated evolution on the human lineage. Characterization of recombination-induced sequence patterns highlights the potential of non-methylation and establishes unmethylated CGIs as targets of meiotic recombination in dogs. These observations describe recombination as an interesting process in genome evolution and provide further insights into the mechanisms of genomic variability.