Domestication has led to genetic changes that affect quantitative traits in farm animals. Both candidate gene analysis using association tests and genome scans based on linkage analysis have been performed to understand the molecular basis underlying quantitative genetic variation in horses, pigs and chickens. To test a possible association of polymorphisms in the PRKAG3 gene, previously found to be associated with excess glycogen content in pig skeletal muscle, with quantitative traits in the horse, the major coding part of the equine PRKAG3 sequence was identified. Bioinformatic characterization of the equine PRKAG3 gene was conducted. A single nucleotide polymorphism (SNP) causing a missense mutation (Pro258Leu) was found. Screening this SNP showed that the Leu258 allele was more frequent in breeds with heavy muscularity. To assess previously reported associations between polymorphisms in the MC4R gene and obesity-related traits further, we conducted linkage analysis between the MC4R locus and fatness-related traits using a Wild BoarxLarge White intercross. No significant association between segregation at the MC4R locus and fatness was detected in this pedigree. A genome scan of quantitative trait loci (QTLs) has been performed in an intercross between chicken lines divergently selected for growth. Divergent parental lines have been established by selecting for high and low 56-day body weight for over 40 generations. The selection has led to approximately a 9-fold difference in 56-day body weight between lines and resulted in correlated responses for a number of traits including appetite, immune response, body composition and metabolic traits. Phenotypic data on growth and other correlated traits were collected from more than 800 F2 individuals. Genome scans using 145 markers on 26 linkage groups have identified QTLs affecting growth and correlated responses to selection for 56-day body weight. No major QTL explaining a large portion of phenotypic variation in growth was revealed in this study.

L’acétylation des résidus de glucosamine terminaux par la N-acétyltransférase lysosomale (HGSNAT) est une étape essentielle de la dégradation catabolique de l’héparan sulfate. Des défauts dans cette réaction causent une maladie de surcharge lysosomale autosomale récessive rare : le désordre de Sanfilippo type C (SFC). À ce jour, 54 mutations ont été rapportées chez des patients SFC, incluant 13 mutations des sites d’épissage, 11 insertions et délétions, 8 mutations non-sens, 18 mutations faux-sens et 4 polymorphismes, avec différentes manifestations phénotypiques. Nous avons identifié 10 d’entre elles et effectué une étude exhaustive portant sur l’éventail des mutations SFC, leur distribution dans la population de patients, ainsi que leur impact potentiel sur la structure de la HGSNAT. Les erreurs d’épissage, les mutations non-sens, les insertions et les délétions devraient toutes entraîner un ARN non fonctionnel qui est rapidement dégradé par des mécanismes de contrôle qualité cellulaire. Les 4 polymorphismes identifiés sont des changements d'acides aminés qui ne modifient pas l'activité enzymatique, la glycosylation ou la localisation et n'ont donc pas de signification au niveau clinique. Au niveau des enzymes, les polymorphismes sont des changements d’acides aminés qui n’affectent pas la fonction, mais dans un contexte d’acides nucléiques ils peuvent être considérés comme des mutations faux-sens. Les dix-huit mutations faux-sens qui ont été exprimées ont produit des protéines inactives, en raison d'erreurs dans leur repliement. Ceci expliquerait donc la progression sévère de la maladie chez les personnes porteuses de ces mutations. Les protéines mutantes mal repliées sont anormalement glycosylées et conservées dans le réticulum endoplasmique. La thérapie par amélioration de l’activité enzymatique par des chaperonnes est une option thérapeutique potentielle, spécifiquement conçue pour exploiter l'activité enzymatique résiduelle de mutants mal repliés, afin d’éliminer les substrats stockés. Nous avons démontré que le traitement de plusieurs lignées de fibroblastes de patients SFC avec le chlorhydrate de glucosamine, un inhibiteur spécifique de la HGSNAT, a partiellement restauré l’activité de l'enzyme mutante, fournissant une preuve de l’utilité future de la thérapie par des chaperonnes dans le traitement de la maladie de SFC. / The acetylation of terminal glucosamine residues by lysosomal N-acetyltransferase (HGSNAT) is an essential part of the catabolic breakdown of heparan sulfate. Defects in this reaction result in the rare autosomal recessive lysosomal storage disorder Sanfilippo syndrome type C (SFC). To date 54 mutations in SFC patients have been reported including 13 splice-site mutations, 11 insertions and deletions, 8 nonsense, 18 missense and 4 polymorphisms, with different phenotypic manifestations. We have identified 10 of them and conducted a comprehensive review discussing the spectrum of Sanfilippo C mutations, their distribution within the patient population as well as how the mutations could potentially affect the structure of HGSNAT. Splicing errors, nonsense mutations, insertions and deletions were all predicted to result in non-functional RNA which is rapidly degraded by cellular quality control mechanisms. The 4 identified polymorphisms resulted in amino acid changes which did not affect the enzyme activity, glycosylation or targeting and were therefore not clinically significant. Polymorphisms, in the context of enzymes are amino acid changes not affecting function, but in the context of nucleic acids can still be considered as missense mutations. Eighteen missense mutations were expressed and shown be inactive due to errors in protein folding providing an explanation for the severe disease progression seen in individuals with these mutations. Misfolded mutants were abnormally glycosylated and retained in the endoplasmic reticulum. Enzyme enhancement/chaperone therapy is a potential treatment option specifically designed to exploit the residual enzyme activity of misfolded mutants in order to clear stored substrates. We demonstrated that treatment of several fibroblast lines of SFC patients with a specific inhibitor of HGSNAT; glucosamine-hydrochloride partially rescued mutant enzyme activity providing a proof of principle for the future use of chaperone therapeutics in the treatment of SFC.

THESIS SUBMITED IN FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF TECHNOLOGY OF BIOMEDICAL TECHNOLOGTY IN THE FACULTY OF HEALTH AND WELLNESS SCIENCES AT THE CAPE PENINSULA UNIVERSITY OF TECHNOLOGY SUPERVISORS: PROF T.E. MATSHA PROF R.T. ERASMUS DR A. ZEMLIN SUBMITED DECEMBER 2013 / Introduction: Cardiovascular diseases (CVD) have become the leading cause of morbidity and mortality amongst the global population. Originally thought to be a health burden of high income countries, the prevalence is rapidly increasing in developing countries. For example, in 2008, an estimated 17.3 million died from CVD, and 80% of these (13.8 mil) were from low to middle income countries. Epidemiological data on CVD in Africa is scanty and of poor quality and national vital registration is available in only 5% of Africa’s 53 countries. Furthermore, data on CVD risk amongst the South African population and specifically the mixed ancestry community is poorly described. The increasing global population of people with CVD has been largely attributed to increasing rates of determinants and risk factors which include obesity, metabolic syndrome (MetS), type 2 diabetes mellitus (DM) and chronic kidney diseases (CKD). The prevalence of DM in South Africa is known to be on the rise with more affected communities being South African Asians followed by coloureds. Aims and objectives: The aim of this study was to determine the CVD risk profile of the Bellville South community during a baseline and three year follow-up study, by assessment of known risk factors, MetS, type 2 DM, obesity and CKD. Methods: Participants for this study were drawn from an urban community of the Bellville South suburb of Cape Town. At baseline (January 2008 and March 2009) 946 individuals aged 16 to 95 participated. All participants received a standardized interview and physical examination during which anthropometric measurements were performed three times and their average used for analysis: weight (kg), height (cm), waist (cm) and hip (cm) circumferences. Body Mass Index (BMI) was calculated as weight per square metre (kg/m2). A blood sample was obtained from all participants after an overnight fast for the determination of biochemical profiles: glucose, glycated haemoglobin, creatinine, total cholesterol, high density lipoprotein cholesterol (HDL-C), triglycerides and low density lipoprotein cholesterol (LDL-C) which was calculated using Friedewald’s formula. Kidney function test was assessed through estimated glomerular filtration rate (eGFR) using the cockcroft-Gault and MDRD equations. Blood pressure was measured according to the World Health Organisation (WHO) guidelines. Participants with no history of doctor diagnosed DM underwent a 75 g oral glucose tolerance test as recommended by the WHO. Metabolic syndrome was determined using JIS, NCEP ATPIII and IDF criteria. The follow-up examination was conducted in 2011 (3 years from vii baseline) using similar procedures. A total of 198 participants formed the follow-up cohort whose measurements were compared to those of the baseline. Finally, the prediction and processes/progression of the risk factors were determined. Results: At both baseline and follow-up studies, females had a higher BMI compared to their male counterparts. The crude prevalence of type 2 DM, including the previously diagnosed type 2 DM was 28.59% (age-adjusted = 33.5%, 95%CI: 30.01 – 36.92), and that of undiagnosed type 2 DM was 17.8% (age-adjusted = 12.4%, 95%CI: 9.8 – 14.8). The overall prevalence of CKD was 28.7% (269) and was higher in females (31.4%) compared to 20.2% in males. MetS was present in 46.5% of the participants. Gender-specific prediction for CVD risk calculated using the 30-year CVD interactive risk calculator showed that high CVD risk was present in normoglycaemic and younger subjects (under 35 years). At follow-up, the cumulative incidence of progression in glucose tolerance status was: 16.2% (32 participants including 11 with new-onset diabetes), and increased in a stepwise fashion with the number of components of MetS. Between baseline and 3-year evaluation glomerular filtration rate (eGFR) increased by 8.7 ml/min (95% confidence interval: 6.9-10.7), reflecting variables trajectories across baseline strata of kidney functions. Conclusion: Given the findings of this study and the estimated increases in the determinants and risk factors of CVD in the mixed ancestry population of South Africa this trend may continue to worsen if current trajectories do not change.

Background: The prevalence of diabetes and obesity has been on the rise for many years and the search for new and effective dietetic solutions aiming at reducing calories, reducing body mass and improving diabetes has been ongoing. Currently, the intermittent fasting diet - the practice of alternating periods of eating and fasting - is gaining popularity. One of them is Time-restricted feeding (TRF), which time-limits energy intake within a defined window of time up to 10 hours per day without necessarily altering diet quality or quantity. A reduction in calorie intake, bodyweight, blood pressure, oxidative stress, inflammation biomarkers and triglycerides are evident with TRF studies conducted so far. Aim: The aim of the thesis is to investigate the effects of time-restricted feeding on glycemic biomarkers in human studies. Methods: A literature study is conducted with six chosen experimental studies which are primarily randomized controlled trials or randomized crossover trials with a TRF window of maximum 10 hours per day and predominantly with participants with overweight/obesity, prediabetes, type 2 diabetes and metabolic syndrome. Results: Compared to either baseline and/or control group, fasting glucose was reduced in 3 out of 6 TRF studies, while fasting insulin was reduced in 3 out of 5 TRF studies and HbA1C was decreased in 1 out of 2 TRF studies. For postprandial response, 1 out of 2 TRF studies found a reduction in glucose and likewise for insulin. Mean glucose levels were reduced in 1 out of 3 TRF studies. Insulin resistance was reduced in 3 out of 4 TRF studies while insulin sensitivity was reduced in the one study measuring this. Beta cell function improved in 2 out of 2 TRF studies compared to the control group or baseline. Conclusion: There are indications that TRF has an effect on glycemic biomarkers and thus potentially being able to reduce the risk and/or improve the treatment of type 2 diabetes. But in order to give a more definite answer more studies need to be conducted. In general, these studies should preferably have more participants and be methodologically stronger when it e.g. comes to the control of the dietary regimen.

Polycystic Ovarian Syndrome (PCOS) is an endocrine disorder that affects women’s menstrual cycles and their levels of androgens (male hormones) and cysts on the ovaries. There is a variety of symptoms that come with this endocrine disorder, but insulin resistance is a hallmark symptom of the disorder. It’s shown that 65-70% of women with PCOS have insulin resistance and hyperinsulinemia, this is in women that are overweight, obese, or lean (Marshall & Dunaif, 2012). A lot of women with PCOS find themselves struggling to lose weight because their excess weight is tied to lifestyle and not properly nourishing their bodies, as well as their imbalanced hormones. The understanding of PCOS being a metabolic disorder led to the investigation of the need for registered dietitian nutritionists on the health team of women with PCOS could change the quality of life in women. We developed a survey based on the literature available on the topic of PCOS, diet/nutrition interventions, and the role of RDNs in the healthcare team of PCOS women of childbearing age. Only childbearing-age women (18-44) that have been diagnosed with PCOS were allowed to complete the survey. The survey was comprised of three sections and was designed to be a needs assessment on the need for registered dietitian-nutritionists to be included in the healthcare team of PCOS women. The questions were designed also show any gaps of knowledge or misconceptions about nutrition that these women may have. Lastly, it was designed to examine if women understand how nutrition relates to the management of their symptoms of PCOS and future disease risks. The data from this survey will show the need for RDNs in the healthcare team of PCOS women, and give us an understanding of nutrition education and intervention that could be developed for future studies. This understanding of how RDNs could play a role in symptom management could lead to a better quality of life in PCOS women.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders (“conditions”) then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40–50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.

Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...

Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...