Introdução: Variações genéticas podem influenciar a relação entre ácidos graxos (AG) do plasma e concentração plasmática de biomarcadores inflamatórios. Objetivo: Verificar a associação entre polimorfismos de nucleotídeo único (SNP) presentes nos genes da proteína C reativa (PCR), fator de necrose tumoral (TNF) e interleucina (IL)-10 e AG do plasma e seus efeitos sobre a concentração plasmática de biomarcadores inflamatórios em um estudo de base populacional ISACapital. Métodos: Foram coletadas informações sociodemográficas, de estilo de vida, de atividade física (IPAQ longo), hábito de fumar e beber, bem como amostras de sangue de 281 indivíduos (20 a 59 anos), oriundos de um estudo de base populacional (ISA-Capital). A partir do plasma, foram determinadas as concentrações de IL1, IL6, IL8, IL10, TNF, IL12p70, adiponectina, PCR, proteína quimiotática para macrófagos solúvel (sMCP)1, molécula de adesão intercelular solúvel (sICAM)1 e molécula de adesão celular vascular solúvel (sVCAM)1 por meio da técnica multiplex de imunoensaio e o perfil de ácidos graxos por cromatografia gasosa. O DNA genômico foi extraído e realizada a genotipagem dos SNP presentes no gene da PCR (rs1205, rs1417938, rs2808630), TNF (rs1799964, rs1799724, rs1800629 e rs361525) e IL10 (rs1800871, rs1800896 e rs1800872) pela ténica TaqMan Open Array. Foi realizada análise multivariada de cluster com base nos 11 biomarcadores inflamatórios, permitindo agrupar os indivíduos em grupo inflamado e cluster não inflamado. Resultados: Os indivíduos que possuíam os genótipos GA+AA do SNP -238 G>A (rs361525) do gene do TNF- apresentaram concentrações plasmáticas de TNF-, IL-1, IL-6, IL-10 e IL-12 aumentadas quando comparados com indivíduos homozigotos dominantes. Além disso, homozigotos recessivos do SNP e/i boundary C>T (rs1554286) do gene da IL-10 apresentaram maior concentração plasmática de IL-1 e de TNF- e menor de MCP-1 em relação aos indivíduos homozigotos dominantes. O grupo inflamado apresentou idade, circunferência da cintura, pressão arterial significantemente maiores que o grupo não inflamado. Em relação aos AG do plasma, o grupo inflamado apresentou concentração plasmática de AG palmítico (C16:0), razões AG saturados (SFA)/AG ômega-6 (n-6) e SFA/ AG poli-insaturados (PUFA) e atividade estimada da enzima estearoil CoA desaturase (SCD) aumentadas e concentrações plasmática de PUFA, n- 6 e AG araquidônico (AA) e atividade estimada da enzima delta-5-dessaturase (D5D) reduzidas em comparação com o grupo não inflamado. Interações SNP-AG plasmáticos estatisticamente significante foram detectadas entre o SNP +1919 A>T (rs1417938) do gene da PCR e C16:1n-7, SFA/n-6 e SFA/PUFA; entre o SNP +3872 G>A (rs1205) do gene da PCR e C16:1n-7; entre o SNP e/i boundary C>T (rs1554286) do gene da IL-10 e atividade estimada da enzima D6D; e entre o SNP -1082 A>G (rs1800896) do gene da IL-10 e C18:0, C14:0 e atividade estimada da enzima D5D. Conclusão: Os SNP analisados possuem associações com biomarcadores inflamatórios e os ácidos graxos plasmáticos palmítico, SFA/n-6, SFA/PUFA, SCD-18 foram associados positivamente com um padrão inflamatório, enquanto PUFA, n-6, ácido araquidônico e D5D foram negativamente associados. Dentre as interações encontradas, o AG palmitoleico e a razão SFA/PUFA interagiram com +1919 A>T (rs1417938), e os AG esteárico e mirístico e D5D com -1082 A>G.. / Introduction: Genetics variation can influence the relation between fatty acids (FA) and inflammatory biomarkers levels. Objective: To verify the association between Single Nucleotide Polymorphisms (SNP) in adiponectin, C-Reactive Protein (CRP), Tumor Necrosis Factor (TNF)- and Interleukin (IL)-10 genes and plasma fatty acids and their effects to a systemic inflammatory pattern at a population-based study. Methods: Sociodemographics information, life style, physical activity (IPAQ long form), smoking and drinking habits, as well as blood samples of 281 individuals (20 years 59 years) participants of a population based study (ISA-Capital). Plasma IL1, IL6, IL8, IL10, TNF, IL12p70, adiponectin, CRP, soluble monocyte chemoattractant protein-1 (sMCP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) levels were measured using a multiplex immunoassay and the fatty acid profile was measured by gas chromatography. The DNA was extracted and genotyping of SNP in CPR gene (rs1205, rs1417938, rs2808630), TNF (rs1799964, rs1799724, rs1800629 e rs361525) and IL10 (rs1800871, rs1800896 e rs1800872) was analyzed by TaqMan Open Array. Multivariate cluster analysis was applied on 11 inflammatory biomarkers, allowing to group individuals in inflammatory (INF) or non-inflammatory (NINF) group. Results: Individuals with GA+AA genotypes of SNP -238 G>A (rs361525) of TNF- gene had higher levels of TNF-, IL-1, IL-6, IL-10 and IL-12 compared to GG genotype. Besides that, recessive homozygous of SNP e/i boundary C>T (rs1554286) of IL-10 gene presented higher level of IL-1 and TNF- and lower level of sMCP-1 in relation to dominant homozygous. The INF group had significantly higher age, waist circumference, blood pressure, and total cholesterol than NINF group. Concerning fatty acid profile, INF group had palmitic acid (C16:0), saturated fatty acid (SFA)/omega-6 (n-6) polyunsaturated fatty acid (PUFA) ratio, SFA/PUFA and estimated enzyme activity of stearoyl-CoA desaturase (SCD) levels higher and PUFA, n-6, arachidonic acid and estimated enzyme activity of delta-5 desaturase (D5D) levels lower than NINF group. Significantly interactions were found between of SNP +1919 A>T (rs1417938) of PCR and C16:1n-7, SFA/n-6 and SFA/PUFA; between SNP +3872 G>A (rs1205) of PCR gene and C16:1n-7; between SNP e/i boundary C>T (rs1554286) of IL-10 and estimated enzyme activity of delta-6 desaturase (D6D); and between SNP -1082 A>G (rs1800896) of IL-10 gene and C18:0, C14:0 and estimated D5D activity. Conclusion: The SNP analyzed have associations with inflammatory biomarkers, and plasma palmitic, SFA/n-6, SFA/PUFA, SCD- 18 were positively associated with inflammatory pattern, while PUFA, n-6, arachidonic acid and D5D were negatively associated. Interactions were founded with palmitoleic and SFA/PUFA with +1919.A>T (rs1417938), and stearic and myristic acids and D5D with 1082 A>G.

Magister Scientiae - MSc (Biotechnology) / The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

<p> </p><p>Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs.</p><p>In this project a new kit, TaqManÒ Sample-to-SNP KitÔ <strong>for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method.</strong></p><p>The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure.</p><p>The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.</p><p> </p>

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disorder characterized by a failure in the mechanism of self-tolerance and production of autoantibodies, potentially affecting any organ in the body. The genetic factors behind the disease have been extensively studied in the past years and to date a list of more than 30 loci have been associated with SLE. However, very little is known about the functional significance of the risk variants. In this thesis, we focused on the analysis of SLE-associated variants in three genes: interferon regulatory factor 5 (IRF5), CD226 and the microRNA 146a. In paper I, we analyzed four polymorphisms in the IRF5 gene in a large set of individuals from different populations. We replicated a strong association of a promoter indel in our meta-analysis, but expression analysis indicated that it is rather another variant, SNP rs10954213 in the poly(A) signal of the gene that is in fact the major contributor to the altered gene expression in leukocytes. In manuscript II, we further characterized the regulation of IRF5 expression, showing that this gene can be up-regulated by estrogen in PBMCs and monocytes, regardless of the genotype, which could to some extent, explain the sex-bias of SLE. In paper III, we investigated the association of CD226 with SLE and the potential functional effect of the associated variants. The genetic analysis showed an association of a three-SNP-haplotype located at the 3’UTR region of the gene. The risk haplotype correlated with lower CD226 protein expression on the surface of cytotoxic and helper T cells, as well as in NK T cells. Reporter assays pointed to rs727088 in the 3’UTR as the main responsible variant for altered gene expression. In paper IV, we described the association of a variant in microRNA miR-146a, involved in the interferon pathway, with SLE in Europeans, which could in addition be correlated with decreased expression of both mature and primary miR-146a in leukocytes. In summary, we have investigated the genetic association of three genes with SLE in a large cohort of individuals and identified variants responsible for functional alterations of these genes, providing further insight into the pathogenesis of SLE.

Background: The reasons why some individuals become IgE-sensitised and allergic are largely unknown, though genetic- and early life environmental factors seem to be of importance. Objective: The overall aim of this thesis was to investigate the relationship between IgE-sensitisation and allergic disease, viral infections, genetic markers and early life cytokines. Results: IgE-sensitised children were found to have reduced numbers of IL-12 producing cord blood mononuclear cells (CBMC), whereas children diagnosed with eczema were found to have reduced numbers of IFN-γ producing CBMC. When dividing the children into early onset of IgE-sensitisation and late onset of IgE-sensitisation we found that the children with an early onset had low numbers of PHA-induced IL-4, IL-12 and IFN-γ secreting CBMC. At the age of two there was a general exacerbation of cytokine responses in the IgE-sensitised children, and the results were similar for the children with early onset IgE-sensitisation. Children with a late onset IgE-sensitisation were more similar to the non-sensitised children, but with a specific increase in the response to cat allergen (IL-4 and IFN-γ). The mothers of IgE-sensitised children, were just as their children, found to have an exaggerated cytokine response as compared to mothers of non-sensitised children. Maternal responses correlated well to the responses seen in the child, though the samples were taken two years after delivery. Cytomegalovirus (CMV) infection in early life was associated to reduced numbers of IL-4, and increased numbers of IFN-γ producing cells at the age of two. No association between CMV seropositivity and IgE-sensitisation was seen. Epstein-Barr virus (EBV) infection, on the other hand, was inversely correlated with IgE –sensitisation, whereas no statistically significant association to cytokine production could be seen. We also showed that the IL12B 1188 C-allele was associated to having a positive skin prick test at the age of two. The rare alleles of the three SNPs investigated (IL12B 1188C, IL12RB1132C and IRF1 1688A) were all associated to low IL-12 production at birth. Conclusions: Our results indicate that allergic diseases are complex traits, and that both the genetic and the cytokine background differ between the different allergic diseases. We can also conclude that the time of onset seem to play a role when investigating IgE-sensitisation, and that perhaps early and late onset IgE-sensitisation have partly different causes. CMV and EBV infection early in life are associated to a protective cytokine profile and to protection from IgE-sensitisation, respectively, again indicating the heterogeneity and the complexity of allergic diseases.

Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs. In this project a new kit, TaqManÒ Sample-to-SNP KitÔ for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method. The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure. The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.

Background: The limited within-breed genetic heterogeneity and an enrichment of disease-predisposing alleles have made the dog a very suitable model for the identification of genes associated with risk for specific diseases. Canine mammary cancer is an example of such a disease. However, the underlying inherited risk factors for canine mammary tumours (CMTs) are still largely unknown. In this study, 52 single nucleotide polymorphisms (SNPs) in ten human cancer-associated genes were genotyped in two different datasets in order to identify genes/alleles associated with the development of CMTs. The first dataset consisted of English Springer Spaniel (ESS) CMT cases and controls. ESS is a dog breed known to be at increased risk of developing CMTs. In the second dataset, dogs from breeds known to have a high frequency of CMTs were compared to dogs from breeds with a lower occurrence of these tumours. Results: We found significant associations to CMT for SNPs and haplotypes in the estrogen receptor 1 (ESR1) gene in the ESS material (best P-Bonf = 0.021). A large number of SNPs, among them several SNPs in ESR1, showed significantly different allele frequencies between the high and low risk breed groups (best P-Bonf = 8.8E-32, best P-BPerm = 0.076). Conclusions: The identification of CMT-associated SNPs in ESR1 in two independent datasets suggests that this gene might be involved in CMT development. These findings also support that CMT may serve as a good model for human breast cancer research.

Colorectal cancer (CRC) is a major source of morbidity and mortality in the Western world. Approximately 15% of all CRCs develop via the mutator pathway, which results from a deficiency of mismatch repair (MMR) system and leads to genome-wide microsatellite instability (MSI). MLH1 promoter hypermethylation accounts for the majority of MSI CRCs. Numerous single nucleotide polymorphisms have been identified in MMR genes, however their functional roles in affecting MMR system, and therefore susceptibility to MSI CRCs, are unknown. This study uses a multidisciplinary approach combining molecular genetics, epigenetics, and epidemiology to examine the contribution of MMR gene polymorphisms in CRC. Among a panel of MMR SNPs examined, the MLH1 (-93G>A) promoter polymorphism (rs1800734) was shown to be associated with increased risk of MSI CRCs in two Canadian populations, Ontario and Newfoundland. Functional studies of the MLH1-93G>A polymorphism indicate that it has weak effects on the core promoter activity, although it dramatically reduces activity of the shorter promoter constructs in a panel of cell lines. Furthermore, MLH1 gene shares a bi-directional promoter with EMP2AIP1 gene, and the MLH1-93G>A polymorphism increases the activity of the reverse, EPM2AIP1 promoter. Examination of alternative role of the MLH1-93G>A polymorphism in MSI-H CRCs led to evaluation of a 500-kilobase pair chromosome 3 region around the MLH1 gene and identification of two additional SNPs, rs749072 and rs13098279, which are in strong linkage disequilibrium with rs1800734. All three SNPs showed strong associations with MLH1 promoter methylation, loss of MLH1 protein expression, and MSI-H CRCs in three populations, Ontario, Newfoundland, and Seattle. Such findings potentially implicate genetic susceptibility to DNA methylation. Logistic regression models for MSI-H versus non-MSI-H CRCs demonstrate that models including MLH1 IHC status and MLH1 promoter methylation status fit the data most parsimoniously in all three populations combined, however, when rs1800734/rs749072/rs13098279 was added to this model, polymorphisms no longer remained significant indicating that the observed associations of these polymorphisms with the MSI-H CRCs occur through their effect on DNA methylation. This study identified a novel mechanism in which common missense alterations may contribute to complex disease.

Colorectal cancer (CRC) is a major source of morbidity and mortality in the Western world. Approximately 15% of all CRCs develop via the mutator pathway, which results from a deficiency of mismatch repair (MMR) system and leads to genome-wide microsatellite instability (MSI). MLH1 promoter hypermethylation accounts for the majority of MSI CRCs. Numerous single nucleotide polymorphisms have been identified in MMR genes, however their functional roles in affecting MMR system, and therefore susceptibility to MSI CRCs, are unknown. This study uses a multidisciplinary approach combining molecular genetics, epigenetics, and epidemiology to examine the contribution of MMR gene polymorphisms in CRC. Among a panel of MMR SNPs examined, the MLH1 (-93G>A) promoter polymorphism (rs1800734) was shown to be associated with increased risk of MSI CRCs in two Canadian populations, Ontario and Newfoundland. Functional studies of the MLH1-93G>A polymorphism indicate that it has weak effects on the core promoter activity, although it dramatically reduces activity of the shorter promoter constructs in a panel of cell lines. Furthermore, MLH1 gene shares a bi-directional promoter with EMP2AIP1 gene, and the MLH1-93G>A polymorphism increases the activity of the reverse, EPM2AIP1 promoter. Examination of alternative role of the MLH1-93G>A polymorphism in MSI-H CRCs led to evaluation of a 500-kilobase pair chromosome 3 region around the MLH1 gene and identification of two additional SNPs, rs749072 and rs13098279, which are in strong linkage disequilibrium with rs1800734. All three SNPs showed strong associations with MLH1 promoter methylation, loss of MLH1 protein expression, and MSI-H CRCs in three populations, Ontario, Newfoundland, and Seattle. Such findings potentially implicate genetic susceptibility to DNA methylation. Logistic regression models for MSI-H versus non-MSI-H CRCs demonstrate that models including MLH1 IHC status and MLH1 promoter methylation status fit the data most parsimoniously in all three populations combined, however, when rs1800734/rs749072/rs13098279 was added to this model, polymorphisms no longer remained significant indicating that the observed associations of these polymorphisms with the MSI-H CRCs occur through their effect on DNA methylation. This study identified a novel mechanism in which common missense alterations may contribute to complex disease.