The development of the metabolic syndrome (MetS) and cardiovascular diseases have been suggested to be influenced more by the quality than the amount of dietary fat. The FA composition of serum lipids may be used as biomarkers of dietary fat quality. FAs can, however, also be endogenously synthesized by lipogenic enzymes such as elongases and desaturases. Three desaturases are important in humans: Stearoyl-CoA-desaturase (SCD), ∆6-desaturase (D6D) and ∆5-desaturase (D5D) and surrogate measures of desaturase activities can be estimated as product-to-precursor FA ratios. In this thesis, we demonstrated that high SCD, D6D and low D5D estimated activities predicted MetS 20 years later, as well as cardiovascular and total mortality during a maximum of 33.7 years. The relation between D5D and MetS was independent of lifestyle and BMI, while the relation between SCD, D6D and MetS was confounded by BMI. Serum proportions of palmitic (16:0), palmitoleic (16:1) and dihomo-γ-linoleic acids were higher and the serum proportion of linoleic acid (LA) lower at baseline in those individuals who developed MetS. Further, LA was inversely related to mortality, while palmitic, palmitoleic and dihomo-γ-linoleic acids were directly associated with mortality. We also demonstrated that a diet rich in saturated fat “induced” a similar serum FA pattern (including estimated desaturase activities) that was associated with MetS, cardiovascular disease and mortality. We also propose that the SCD ratio [16:1/16:0] might be a novel and useful marker of dietary saturated fat, at least in Western high-fat diets. Finally, genetic variations in the human SCD1 gene were linked to obesity and insulin sensitivity, results that agree with data in SCD1 deficient mice. This thesis suggests that dietary fat quality and endogenous desaturation may play a role in the development of metabolic and cardiovascular diseases and the results support current dietary guidelines.

Advancements in the field of biotechnology are expanding the scientific horizon and a promising era is envisioned with personalized medicine for improved health. The amount of genetic data is growing at an ever-escalating pace due to the availability of novel technologies that allow massively parallel sequencing and whole-genome genotyping, that are supported by the advancements in computer science and information technologies. As the amount of information stored in databases throughout the world is growing and our knowledge deepens, genetic signatures with significant importance are discovered. The surface of such a set in the data mining process may include causative- or marker single nucleotide polymorphisms (SNPs), revealing predisposition to disease, or gene expression signatures, profiling a pathological state. When targeting a reduced set of signatures in a large number of samples for diagnostic- or fine-mapping purposes, efficient interrogation and scoring require appropriate preparations. These needs are met by miniaturized and parallelized platforms that allow a low sample and template consumption. This doctoral thesis describes an attempt to tackle some of these challenges by the design and implementation of a novel assay denoted Trinucleotide Threading (TnT). The method permits multiplex amplification of a medium size set of specific loci and was adapted to genotyping, gene expression profiling and digital allelotyping. Utilizing a reduced number of nucleotides permits specific amplification of targeted loci while preventing the generation of spurious amplification products. This method was applied to genotype 96 individuals for 75 SNPs. In addition, the accuracy of genotyping from minute amounts of genomic DNA was confirmed. This procedure was performed using a robotic workstation running custom-made scripts and a software tool was implemented to facilitate the assay design. Furthermore, a statistical model was derived from the molecular principles of the genotyping assay and an Expectation-Maximization algorithm was chosen to automatically call the generated genotypes. The TnT approach was also adapted to profiling signature gene sets for the Swedish Human Protein Atlas Program. Here 18 protein epitope signature tags (PrESTs) were targeted in eight different cell lines employed in the program and the results demonstrated high concordance rates with real-time PCR approaches. Finally, an assay for digital estimation of allele frequencies in large cohorts was set up by combining the TnT approach with a second-generation sequencing system. Allelotyping was performed by targeting 147 polymorphic loci in a genomic pool of 462 individuals. Subsequent interrogation was carried out on a state-of-the-art massively parallelized Pyrosequencing instrument. The experiment generated more than 200,000 reads and with bioinformatic support, clonally amplified fragments and the corresponding sequence reads were converted to a precise set of allele frequencies. / QC 20100813

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides. / QC 20101008

ΜΕΛΕΤΗ Α΄ Στόχος: Να διερευνηθεί αν ο κινητικός υπότυπος της αστάθειας κορμού και δυσχέρειας της βάδισης (ΑΚΔΒ) σχετίζεται με τη γνωστική δυσλειτουργία που εμφανίζουν οι ασθενείς με νόσο του Parkinson (NP) χωρίς άνοια. Μέθοδοι: Χορηγήσαμε μια συστοιχία επιλεγμένων νευροψυχολογικών δοκιμασιών σε δύο ομάδες μη ανοϊκών ασθενών με ήπια έως μέτριας βαρύτητας νόσο κατηγοριοποιημένους είτε στον υπότυπο της ΑΚΔΒ είτε σε υπότυπο μη ΑΚΔΒ, καθώς και σε μια ομάδα υγιών μαρτύρων. Οι ομάδες εξισώθηκαν κατά το δυνατόν όσον αφορά δυνητικούς συγχυτικούς παράγοντες που επηρεάζουν τις νευροψυχολογικές επιδόσεις. Αποτελέσματα: Δε διαπιστώθηκαν σημαντικές διαφορές μεταξύ των δύο ομάδων ασθενών στην επίδοση σε οποιαδήποτε από τις χορηγηθείσες νευροψυχολογικές δοκιμασίες. Παρόλα αυτά, σε σχέση με τους μάρτυρες υπήρξε μια τάση διαφοροποίησης ως προς το κυρίαρχο πρότυπο της γνωστικής δυσλειτουργίας. Η ομάδα με τον υπότυπο της ΑΚΔΒ είχε βραδύτερες επιδόσεις σε μια δοκιμασία ψυχοκινητικής ταχύτητας και γνωστικής ευελιξίας, ενώ η ομάδα με υπότυπο της νόσου μη ΑΚΔΒ είχε χειρότερες επιδόσεις στις μετρήσεις της λεκτικής μάθησης και της οπτικοχωρικής αντίληψης. Συμπεράσματα: Ο υπότυπος της ΑΚΔΒ δε συσχετίσθηκε με σοβαρότερα γνωστικά ελλείμματα και έτσι είναι πιθανό οι μηχανισμοί της γνωστικής δυσλειτουργίας να είναι, έως ένα ορισμένο βαθμό, κοινοί ανεξάρτητα από τον κινητικό υπότυπο της νόσου. ΜΕΛΕΤΗ Β Στόχος: Να διερευνηθεί αν υπάρχει συσχέτιση μεταξύ της ΝΡ και του γονιδίου CHRNA4, το οποίο κωδικοποιεί την α4 υπομονάδα του α4β2 νικοτινικού υποδοχέα της ακετυλοχολίνης (nAChR). Mέθοδοι: Στη μελέτη συμμετείχαν 100 ασθενείς με ΝΡ και 105 μάρτυρες, εξισωμένοι ως προς την ηλικία και το φύλο και ανήκοντες στην ίδια πληθυσμιακή ομάδα με τους ασθενείς. Ο γενετικός δείκτης που εξετάσθηκε είναι ένας μονονουκλεοτιδικός πολυμορφισμός στο 5ο εξόνιο του γονιδίου CHRNA4 (dbSNP rs1044396). Έγινε απομόνωση DNA γονιδιώματος από περιφερικό αίμα και ακολούθησε ανάλυση μεγέθους περιοριστικών τμημάτων μετά από αλυσωτή αντίδραση πολυμεράσης και κατάτμηση των προϊόντων αυτής με το ένζυμο Hha I. Μια υποομάδα 42 ασθενών υποβλήθηκαν επίσης σε λεπτομερή κλινική και νευροψυχολογική εκτίμηση. Η στατιστική ανάλυση για τη σύγκριση της συχνότητας των αλληλομόρφων και των γονοτύπων μεταξύ των ομάδων έγινε με τη δοκιμασία χ2, και τον ακριβή έλεγχο Fisher εάν έστω ένα κελί είχε n<5. Υπολογίστηκαν οι σχετικοί κίνδυνοι και τα κατά 95% διαστήματα αξιοπιστίας τους που αντιστοιχούσαν στα αλληλόμορφα και τους γονότυπους. Χρησιμοποιήθηκε η λογιστική ανάλυση παλινδρόμησης εάν ήταν απαραίτητη η προσαρμογή για την ηλικία ή το φύλο. Αποτελέσματα: Οι συχνότητες των γονοτύπων στην ομάδα των ασθενών (TT 34%; CT 58%; CC 8%) σε σύγκριση με τις συχνότητες των γονοτύπων στην ομάδα των μαρτύρων (TT 28.6 %; CT 47.6%; CC 23.8 %) παρουσίασαν στατιστικά σημαντική διαφορά (χ2 = 9.48, df = 2, p = 0.009). Η ομοζυγωτία CC συσχετίσθηκε με χαμηλότερο κίνδυνο παρουσίας της ΝΡ (CC vs φορείς T: OR = 0.28; 95% CI = 0.12–0.65; p = 0.002; στατιστική ισχύς 93.1%). Παρατηρήθηκε επίσης απόκλιση στην κατανομή των αλληλομόρφων μεταξύ των ασθενών και των μαρτύρων. Υπήρχε σημαντικά χαμηλότερη συχνότητα του αλληλόμορφου C μεταξύ των ασθενών (37%) σε σχέση με τους μάρτυρες (47.6%) (χ2 = 4.73; df = 1; OR=0.65; 95% CI = 0.44–0.96; p = 0.03). Η ανάλυση διαστρωμάτωσης έδειξε ότι η διαφορά στην κατανομή των γονοτύπων μεταξύ ασθενών και μαρτύρων ήταν στατιστικά σημαντική και συγκριτικά μεγαλύτερη στο θήλυ φύλο σε σχέση με το άρρεν φύλο και στους ασθενείς με εκδήλωση ΝΡ σε όψιμη ηλικία ( > 50 ετών) σε σχέση με αυτούς που εμφάνισαν πρώιμης έναρξης νόσο (< 50 ετών). Οι ασθενείς με ΝP που ανιχνεύθηκαν να φέρουν το γονότυπο CC και υποβλήθηκαν σε νευροψυχολογική αξιολόγηση έτειναν να έχουν καλύτερα διατηρημένες τις γνωστικές λειτουργίες που σχετίζονται με την προσοχή και την ταχύτητα επεξεργασίας των πληροφοριών. Συμπεράσματα: Η παρουσία του αλληλομόρφου C (dbSNP rs1044396) του γονιδίου CHRNA4 συνδέεται με μειωμένο κίνδυνο ΝΡ κατά 35%. Επίσης, τα άτομα με το γονότυπο CC εμφανίζουν σχεδόν τρισήμισυ (3,5) φορές χαμηλότερο κίνδυνο νόσου του Parkinson. Η ποικιλομορφία του γονιδίου CHRNA4 φαίνεται ότι σχετίζεται ιδιαίτερα με την επιρρέπεια εκδήλωσης της ΝΡ με ηλικιακά όψιμη έναρξη της νόσου, και επίσης με τις γνωστικές λειτουργίες των ασθενών χωρίς άνοια, ειδικά αυτές που εξαρτώνται από την προσοχή και την οπτικοκινητική αντίληψη. / Study A Aim: To investigate whether there is an association of the postural instability and gait difficulty (PIGD) motor subtype with cognitive dysfunction in non-demented Parkinson’s disease (PD) patients. Methods: We administered a battery of selected neuropsychological tests to assess attention, psychomotor speed, executive functions (set shifting ability and inhibitory control), visuospatial perception and visual constructive ability to two groups of non-demented patients with mild to moderate disease classified either as PIGD or as non-PIGD subtype and to a group of healthy controls. Groups were matched on potential confounders of neuropsychological performance. Results: No significant differences were revealed between the two groups of patients in the performance of any of the administered neuropsychological tests. However, relative to controls there was a tendency towards a differential pattern of cognitive dysfunction. The PIGD group had slower performance in a test of psychomotor speed and cognitive flexibility, whilst the non-PIGD group performed worse in measures of verbal learning and visuo-spatial perception. Conclusions: The PIGD subtype was not associated with more severe cognitive deficits and may to a certain extent share common mechanisms of cognitive dysfunction with non-PIGD subtypes. Study B Aim: to investigate whether there is an association between PD and a variation in the CHRNA4 gene coding for the α4 subunit, the primary subunit of the α4β2 brain nicotinic acetylcholine receptors. Methods: Patients (N=100) and controls (N=105), matched on the basis of sex, age and ethnicity, were genotyped for a single nucleotide polymorphism at cDNA position 1860 lying within the 5th exon of the CHRNA4 gene. DNA was extracted from peripheral blood samples and genotyping was done by PCR-based restriction fragment length polymorphism analysis. A subset of 42 patients also received detailed clinical and cognitive assessments. Comparisons of allele and genotype frequencies between groups were performed using the χ2 test, and the Fisher exact test if one cell had n<5. The relative risk for genotypes and alleles was estimated through calculation of odds ratios (ORs) with 95% confidence intervals (CIs). Logistic regression analysis was used if adjustment for age or sex was necessary. Results: The genotype frequencies in the patients group (TT 34%; CT 58%; CC 8%) vs. the genotype frequencies in the control group (TT 28.6 %; CT 47.6%; CC 23.8 %) demonstrated a statistically significant difference (χ2 = 9.48, df = 2, p = 0.009). CC homozygosity was associated with a lower risk of PD (CC vs T carriers: OR = 0.28; 95% CI = 0.12–0.65; p = 0.002). Also, the allelic distribution was significantly different between patients and controls. There was a significantly lower frequency of the C allele among the patients with PD (37%) as compared with the controls (47.6%) (χ2 = 4.73; df = 1; OR=0.65; 95% CI = 0.44–0.96; p = 0.03). Stratified analysis showed that the difference in the genotypic distribution between cases and controls was significant among females but did not reach significance among males. The frequency of CC homozygotes was also significantly lower in the group of patients with late onset PD than in the controls, but it was not significantly different between the early onset group of patients and the controls. CC homozygotes also tended to have better performance than T carriers on measures of attention and psychomotor speed (Trail Making Test part A and Symbol Digit Modalities Test). Conclusions: The presence of the C allele at SNP rs1044396 of the CHRNA4 gene is associated with a decreased risk for PD by 35%. Moreover, the CC genotype lowers the risk for PD by ~ 3.5 fold. Variation in the CHRNA4 gene may particularly influence susceptibility for late onset PD and further be associated with measurable effects on overt cognitive performance of yet not-demented PD patients, specifically the part loading on attentional capacities.

No description available.

No description available.

L’immunité adaptive et la discrimination entre le soi et le non-soi chez les vertébrés à mâchoire reposent sur la présentation de peptides par les récepteurs d’histocompatibilité majeur de classe I. Les peptides antigéniques, présentés par les molécules du complexe d’histocompatibilité (CMH), sont scrutés par les lymphocytes T CD8 pour une réponse immunitaire appropriée. Le répertoire des peptides du CMH de classe I, aussi appelé immunopeptidome, est généré par la dégradation protéosomale des protéines endogènes, et a un rôle essentiel dans la régulation de l’immunité cellulaire. La composition de l’immunopeptidome dépend du type de cellule et peut présenter des caractéristiques liées à des maladies comme le cancer. Les peptides antigéniques peuvent être utilisés à des fins immunothérapeutiques notamment dans le traitement voire la prévention de certains cancers. La spectrométrie de masse est un outil de choix pour l’identification, le séquençage et la caractérisation de ces peptides. Cependant, la composition en acides aminés, la faible abondance et la diversité de ces peptides compliquent leur détection et leur séquençage. Nous avons développé un programme appelé StatPeaks qui permet de calculer un certains nombres de statistiques relatives à la fragmentation des peptides. À l’aide de ce programme, nous montrons sans équivoque que les peptides du CMH classe I, en mode de fragmentation par dissociation induite par collision (CID), fragmentent très différemment des peptides trypsiques communément utilisés en protéomique. Néanmoins, la fragmentation par décomposition induite par collision à plus haute énergie (HCD) proposée par le spectromètre LTQ-Orbitrap Velos améliore la fragmentation et fournit une haute résolution qui permet d’obtenir une meilleure confiance dans l’identification des peptides du CMH de classe I. Cet avantage permet d’effectuer le séquençage de novo pour identifier les variants polymorphes qui ne sont normalement pas identifiés par les recherches utilisant des bases de données. La comparaison des programmes de séquençage Lutefisk, pepNovo, pNovo, Vonode et Peaks met en évidence que le dernier permet d’identifier un plus grand nombre de peptides du CMH de classe I. Ce programme est intégré dans une chaîne de traitement de recherche d’antigènes mineurs d’histocompatibilité. Enfin, une base de données contenant les informations spectrales de plusieurs centaines de peptides du CMH de classe I accessible par Internet a été développée. / Adaptive immunity and discrimination between self and nonself in jawed vertebrates relies on the presentation of peptides by the major histocompatibility (MHC) class I receptors. Foreign or self peptide antigens presented by the MHC molecules are probed by CD8 T-cell lymphocyte for proper immune response. The repertoire of MHC I peptides collectively referred to as the immunopeptidome is generated through the proteasomal degradation of endogenous proteins and plays an important role in the regulation of cellular immunity. The composition of the immunopeptidome is cell specific and can harbor important hallmark of human diseases including cancer. Antigenic peptides can also be used in immunotherapy to mount an appropriate immune response against cancer cells displaying these peptides. Mass spectrometry is a tool of choice for the identification, sequencing and characterization of these peptides. However, the amino acid composition, the low abundance and diversity of these peptides make their detection and sequencing more challenging. We developed a software, called StatPeaks, that calculates statistics relative to the fragmentation of peptides. Using this software, we demonstrate that under collision induced dissociation (CID) MHC class I peptides fragment in a very different fashion than tryptic peptides, commonly used in proteomics. However, the higher-energy collisional dissociation (HCD) mode available on the LTQ-Orbitrap Velos enhances peptide fragmentation and provides high resolution fragment information that significantly improves the confidence in MHC class I peptide identification. This inherent advantage confers the ability to perform de novo sequencing to identify polymorphic variants that would normally elude conventional database searches. The comparison of de novo peptide sequencing software Lutefisk, pepNovo, pNovo, Vonode and Peaks indicated that the later software enabled higher rates of correct identification for MHC class I peptides. This software was integrated into a data analysis pipeline for the identification minor histocompatibility antigens (MiHAs). A web-based library that stores spectral information of hundreds of synthetic MHC class I peptides was developed in support to the needs of the immunopeptidome discovery program.

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Metachromatic leukodystrophy (MLD) and Gaucher disease (GD) are caused by a deficiency of arylsulphatase A (ASA) and b-glucocerebrosidase (GBA), respectively. They are lysosomal storage disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement resulting in high morbidity and mortality. The overall aim of this study is to elucidate the genetic component/s of high ASA and GBA enzyme activity in normal healthy individuals with the ultimate goal of using this information to produce greater protein activity from a recombinant protein. A wide variation in ASA and GBA enzyme activity levels has been observed in the normal population. The first objective of this project was to identify and characterise single nucleotide polymorphisms (SNPs) in the arylsulphatase A (ARSA) and glucocerebrosidase (GBA) genes that are responsible for determining the levels of expressed enzyme activity in the normal population. The second objective was to assess the contribution of transcriptional regulation and TCP80 mediated translational control to normal enzyme variation. TCP80, a translational control protein that interacts with the GBA coding region, is a splice variant of the interleukin binding factor 3 (ILF3) gene. Ten samples from individuals with high ASA activity and twenty samples from individuals with high GBA activity were screened for polymorphisms via denaturing high pressure liquid chromatography (dHPLC) and sequencing. The frequency of these polymorphisms in the normal population was determined using dot-blot hybridisation. Fifteen ARSA polymorphisms (4 promoter, 5 coding, 5 intronic and 1 poly(A) signal) and two GBA polymorphisms (1 intronic and 1 in 3¢-UTR) were identified. Two low frequency ASA polymorphisms (2723A > G, W193C) were found to be correlated with low activity, while another low frequency ASA polymorphism (1101+123C > T) was found to be correlated with high activity in a population of 113 individuals. Real time PCR was used to measure mRNA levels of GBA, ASA and LF3 along with enzyme activity levels of GBA and ASA in two cell types (leucocytes and skin fibroblasts) from four healthy individuals and seven cell lines (HL60, THP1, Huh7, U118, SW1353, Hep G2, and B-cells). Transcriptional control was evident for all three genes with GBA mRNA levels varying over 30 fold, ASA mRNA levels varying over seven fold and ILF3 levels varying more than 24 fold. The 5¢-flanking region of GBA was investigated for the cis-elements responsible for tissue-specific expression. However, it was not possible to demonstrate that the cis-element region was influencing GBA expression. Translational efficiency was measured using the magnitude of the mRNA:enzyme activity ratio as an indicator. GBA translational inefficiency was most pronounced in B cells which require four times more mRNA molecules than hepatocytes (Hep G2) and over 25 times more mRNA molecules than chondrocytes (SW1353) to produce one unit of GBA enzyme activity. Except in B-cells, GBA translational efficiency appears to increase as ILF3 mRNA levels decrease. The tissue-specific variation observed in the protein levels of the ILF3 splice variants, TCP80 and DRBP76, may play a role. The correlation of several low frequency SNPs with low ASA enzyme activity or high ASA activity indicates a role in determining the distribution of enzyme activity levels in the normal population. However, there do not appear to be any common high activity polymorphisms. Knowledge of the exact mechanisms responsible for the observed transcriptional and translational control of these lysosomal genes will greatly enhance the understanding of genotype-phenotype correlation and the contribution of genetic variants to natural variation.