Dissertations / Theses on the topic 'Obesity – Genetic aspects'

Obesity is a complex disorder which has reached epidemic proportions in many parts of the world. Twin studies have demonstrated a high heritability for obesity. The subsequent appli-cation of genome wide association studies (GWAS) in the last decade have identified at least 32 genetic loci associated with body mass and obesity. Despite these great advances, these loci are almost exclusively completely naïve in a functional context. Genetic variations within the gene encoding the fat mass and obesity associated gene (FTO) are the strongest and most consistently observed genetic variants associated with obesity and body mass throughout various studied populations from all parts of the world. The identification of association of FTO with obesity has spurred immense interest in the function of the FTO protein and the functional consequences of its variants. However, the implications of genetic variants at other genetic loci on protein molecular function and body mass development remain undetermined. This thesis aims to examine more closely four of the genetic loci associated with obesity; in proximity of, or associated with: FTO, TMEM18, MAP2K5 and STK33, in two cohorts of children of European descent: a case-control of clinically obese children and normal weight controls from the Stockholm area; and a cross sectional cohort of Greek children. These smaller cohorts allow for studies of more specific effects of genetic variants as individuals in these cohorts can be more carefully studied. TMEM18 gene expression was also studied in the rat-brain where a positive correlation was observed between the body weight of the animal and TMEM18 expression. We also employed next generation sequencing to more carefully study obesity-associated genetic loci related to FTO and TMEM18. We utilized a novel strategy in this project to study genetic variation in the entire FTO- and TMEM18 genes, as well as in the GWAS-identified BMI-associated loci located downstream from TMEM18. This analysis was performed on a case-control cohort of Swedish children (n = ~1000). Through this analysis, we were able to observe genetic variants within intron 1 of the FTO gene to be the main genetic variants asso-ciated with obesity at this locus. We also observed, for the first time, obesity-associated genetic variants within the gene encoding TMEM18. To analyze the potential functional context of FTO we used an in silico approach, utilizing public information databases on mRNA co-expression and protein-protein interaction. Based on our findings, we speculate on a wider functional role of FTO in extracellular ligand-induced neuronal plasticity, possibly via interaction or modulation of the BDNF/NTRK2 signaling pathway.

The current epidemic of obesity poses a substantial threat to public health worldwide. Obesity is associated with many deleterious health conditions, including type 2 diabetes, hypertension, dyslipidaemia, respiratory conditions, arthritis, and some forms of cancer. Moreover, the rising prevalence of obesity has been accompanied by a substantial increase in the cost of treating these conditions. Obesity results from a complex interaction between behavioural, environmental, and genetic factors. While the recent increase in the prevalence of obesity is largely due to behavioural factors (for example, physical inactivity); it has also been observed that genetic factors make a large contribution to individual susceptibility. In fact, studies indicate that as much as 50 - 80% of the variation in measures of obesity can be attributed to the effects of genes. Furthermore, closer examination of this genetic component using segregation analysis has indicated the presence of common genes for obesity, with large effects on the phenotype. However, these putative major genes for obesity have not yet been identified. The aim of this thesis was to investigate the role of three distinct genetic loci in obesity and related cardiovascular factors, including type 2 diabetes and dyslipidaemia. The aim of the first investigation was to test whether a common polymorphism (Pro12Ala) in the gene encoding peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) was associated with obesity and other cardiovascular risk factors in a large group of Caucasian subjects. PPAR-γ2 is an adipogenic transcription factor, which also regulates insulin sensitivity in adipose tissue. No association was observed between the Pro12Ala polymorphism and obesity in Caucasians, but obese subjects carrying the Ala allele displayed an altered blood lipid profile compared with obese Pro/Pro subjects. As the Pro12Ala polymorphism may exacerbate the risk of cardiovascular disease by modifying blood lipid profile in obesity, this relationship was examined further in a separate population. The aim of the second investigation was to determine whether the Pro12Ala polymorphism was associated with obesity, dyslipidaemia, diabetes and carotid intima-medial wall thickening in a population at high risk of developing cardiovascular disease. Australian Aboriginal people display high rates of mortality from cardiovascular disease, and it is possible that their increased susceptibility is due to genetic factors. However, the results from the Aboriginal population confirmed the results of the first study: there was no intrinsic association between the Pro12Ala variant and obesity. In addition, the Ala allele was not associated with deleterious changes in blood lipid profile, as it was in Caucasians. The aim of the third investigation was to confirm the presence of a quantitative trait locus (QTL) for obesity on chromosome 20q13. Highly polymorphic genetic markers in this region were tested for linkage and association with several measures of obesity in a Caucasian population. None of the measures of obesity were linked to or associated with markers spanning 20q13, suggesting that this chromosomal region does not contain a major locus for obesity in this Caucasian population. In the fourth investigation, the 5' sequence of Agouti Signalling Protein (ASIP) was identified. ASIP is a candidate gene for obesity, as it is expressed at high levels in adipocytes, and may participate in several obesity-related processes. Three new exons and two alternative promoters were identified for the ASIP gene. These results may lead to greater understanding of the role of ASIP in obesity and adipocyte metabolism; and may also be used to direct further research into genetic variation within this candidate gene. In conclusion, extensive study of two established candidate genetic loci revealed no association with measures of obesity. Therefore, it is likely that loci other than these make significant contributions to obesity in humans. Further investigation of novel candidate genes, such as ASIP, may allow the identification of novel genetic polymorphisms and new pathways important for the genetic basis of obesity.

Background and objectives: Recent genome-wide association (GWA) studies conducted in Caucasian populations have significantly expanded the list of confirmed and potential susceptibility genes for obesity and type 2 diabetes. The major objective of this thesis was to establish the role of the previously identified obesity- and T2DM-susceptibility genes in the Hong Kong Southern Chinese population. Major findings: In a cross-sectional case-control study of Southern Chinese which involved 470 obese cases and 700 normal-weight controls, significant associations with obesity were demonstrated in 7 of 13 single nucleotide polymorphisms (SNPs) that have shown significant associations with obesity and/or body mass index (BMI) in previous Caucasian GWA studies. These SNPs are located within or near the GNPDA2, FTO, MC4R, KCTD15, SFRS10-ETV5-DGKG, SEC16B-RASAL2 and NEGR1 loci. The combined genetic risk score (GRS) of the 13 studied SNPs was associated with an increased risk for obesity. The GNPDA2 rs10938397, FTO rs8050136, and MC4R rs17782313, which showed the most significant associations with obesity, were further examined for their associations with persistent central obesity and the metabolic syndrome (MetS). Both rs8050136 and rs10938397 were significantly associated with persistent central obesity. rs10938397 was also associated with the MetS. The combined GRS of these 3 SNPs showed significant associations with both persistent central obesity and persistent MetS. Nineteen multimarker-tagging SNPs that span a well-defined LD block of the FTO gene were evaluated for their associations with obesity in a case-control study which involved 249 cases and 400 controls. rs16952522 was found to be significantly associated with obesity, in addition to the well-known SNP rs8050136. These 2 SNPs were nominally associated with T2DM, although the associations were abolished after adjustment for age, sex and BMI. However, the GA haplotype composed of the risk alleles of these 2 SNPs was significantly associated with T2DM, independent of BMI. Seventeen previously identified T2DM-associated SNPs were investigated for the associations with glycaemic progression in an 8-year follow-up study which involved 518 cases and 998 controls. Their combined GRS was associated with an increased risk for glycaemic progression. A significant association with glycaemic progression was found with CDKN2A/B rs10811661. Moreover, KCNJ11 rs5219 and IGF2BP2 rs11711477 also showed potential associations with glycaemic progression. In the subsequent 12-year follow-up study, which involved 200 cases and 903 controls, the CDKN2A/B rs10811661 showed a significant independent association with incident T2DM. The KCNJ11 E23K (rs5219) variant was examined for its association with diabetes development in a 12-year prospective study. It was found to be significantly associated with the development of prediabetes but not with the development of T2DM. However, in a meta-analysis which involved 15680 subjects across different populations, this variant could indeed predict T2DM. Conclusions: The findings of this thesis have provided novel evidence supporting the role of the GWA studies-identified obesity- and T2DM-associated genetic variants as genetic markers of obesity and T2DM among Southern Chinese in Hong Kong, and suggest that the GNPDA2, FTO and MC4R genes confer susceptibility to obesity and that the CDKN2A/B and KCNJ11 genes may play a role in diabetes development.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Obesity is a disease characterised by an excess of white adipose tissue. It leads to increased risk of several associated disorders including cardiovascular disease and type 2 diabetes mellitus. The number of people who are obese is increasing worldwide and as such much research is conducted on obesity and associated disorders. The use of animal models in the study of obesity allows understanding of involvement of specific genes in fat formation and related processes. This can be used to identify targets for prevention or treatment of obesity. Kriippel—like factors are a family of transcription factors that have been implicated in a number of roles, one of which is metabolism. The Klf3'/' mouse has decreased adipose tissue but the metabolic implications of this had not been determined. Klf8 has high homology to Klf3 in the zinc finger region and was suspected to have a role in metabolism and fat formation. A metabolic role for Klf8 had not been investigated. In this thesis, the metabolic profiles of Klf3— and Klf8—deficient mice were examined. Klf3'/' male mice were found to be resistant to diet-induced weight gain. They maintained lower WAT mass and smaller adipocytes. In addition, these mice had reduced hepatic steatosis and improved glucose tolerance and circulating hormone levels. Klf88W male and Klf8+/g’ female mice had no clear phenotype suggesting Klf8 either has roles in areas other than metabolism, or that functional redundancy occurs, thus compensating for the decreased Klf8. These studies show that Klf3 could potentially be targeted for treatments of obesity as mice lacking this protein are lean, resistant to diet-induced obesity and appear metabolically healthy.

Pomegranate flower (PGF) has been prescribed in Unani and Ayurvedic medicines for the treatment of diabetes. It has been demonstrated that PGF extract activates peroxisome proliferator-activated receptor (PPAR) -or and -y to improve glucose tolerance, hyperlipidemia and excess cardiac triglyceride (TG) accumulation in Zucker diabetic fatty (ZDF) rats. Fatty liver (hepatic steatosis), the most common cause of abnormal liver function tests, is strongly associated with insulin resistance. The effect and its underlying mechanism of PGF on fatty liver was investigated in ZDF rats and in human liver-derived HepG2 cell line. Male ZDF rats were treated with PGF extract (500 mg/kg, once daily) for 6 weeks. At the end of treatment, liver weight index was measured. Hepatic contents of TG and total cholesterol (TC) were measured using enzymatic colorimetric methods. Fat droplet accumulations in the livers were determined with an Oil Red 0 staining method. Hepatic gene profiles were analyzed by RT—PCR. PGF treatment reduced the ratio of liver weight to tibia length (an indicator of hepatomegaly), hepatic TG content and lipid droplets in ZDF rats, compared to untreated animals. These effects were accompanied by enhanced hepatic mRNA expression of PPAR-oc, carnitine palmitoyltransferase-l and acyl-CoA oxidase, and reduced stearoyl-CoA desaturase-l. In contrast, PGF showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and TG. Furthermore, PGF extract treatment enhanced endogenous expression of PPAR-oc and acyl-COA oxidase mRNAs in HepG2 cell line. Thus, my present findings suggest that PGF extract ameliorates fatty liver in ZDF rats, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation. This study provides insights into the mechanism of the unique traditional medicine in improving insulin resistance. Moreover, these findings are potentially important for supporting the extension of these findings to clinical trials to demonstrate the effectiveness of PGF in the prevention and/or treatment of diabetes and obesity-induced non-alcoholic fatty liver disease, through modulation of abnormal lipid metabolism.

Obesity is a common disease among all ages that has threatened human health and has become a global concern. Gut microbiota can affect human metabolism and thus may modulate obesity. Certain mixes of gut microbiota can protect the host to be healthy or predispose the host to obesity. Modern next-generation sequencing technique allows accessing huge amount of genetic information underlying microbiota and thus provides new insights into the functionality of these micro-organisms and their interactions with the host. Multiple previous studies have demonstrated that the microbiome might contribute to obesity by increasing dietary energy harvest, promoting fat deposition and triggering systemic inflammation. However, these researches are either based on lab cultivation studies or basic statistical analysis. In order to further explore how gut microbiota affect obesity, this thesis utilize a series of machine learning methods to analyze large amount of metagenomics data from human gut microbiome. The publicly available HMP (Human Microbiome Project) metagenomic sequencing data, contain microbiome data for healthy adults, including overweight and obese individuals, were used for this study. HMP gut data were organized based on two different feature definitions: taxonomic information and metabolic reconstruction information. Several widely used classification algorithms: namely Naive Bayes, Random Forest, SVM and elastic net logistic regression were applied to predict healthy or obese status of the subjects based on the cross-validation accuracy. Furthermore, the corresponding feature selection algorithms were used to identify signature features in each dataset that lead to the differences between healthy and obese samples. The results showed that these algorithms perform poorly on taxonomic data than metabolic pathway data though lots of selected taxa are still supported by literature. Among all the combinations between different algorithms and data, elastic net logistic regression has the best cross-validation performance and thus becomes the best model. In this model, several important features are found and some of these are consistent with the previous studies. Rerunning classifiers by using features selected by elastic net logistic regression again further improved the performance of the classifiers. On the other hand, this study uncovered some new features that haven't been supported by previous studies. The new features could also be the potential target to distinguish obese and healthy subjects. The present thesis work compares the strengths and weaknesses of different machine learning techniques with different types of features originating from the same metagenomics data. The features selected by these models could provide a deep understanding of the metabolic mechanisms of micro-organisms. It is therefore worth to comprehensively understand the differences of gut microbiota between healthy and obese subjects, and particularly how gut microbiome affects obesity.

Thesis (PhD)--Stellenbosch University, 2012.
Includes bibliography
ENGLISH ABSTRACT: Background/Aims: Obesity has increased rapidly in South African children and adolescents with significant variability observed among racial groups. Genes that regulate appetite have been studied in different populations worldwide, but their role in obesity among South African adolescents is unknown. The present study aimed at investigating the role of these genes, and their combined effect with physical activity in the development of obesity among South African adolescents. Methods: A total of 1564 South African school learners of Caucasian (n= 146), Mixed Ancestry (n= 872) and Black African (n= 537) ethnic groups were recruited for a research project that aimed to elucidate diabetes and the metabolic syndrome in children and adolescents attending schools in periurban areas of the Western Cape. The present case-control study included 227 obese-overweight (115 Black Africans and 112 Mixed Ancestry), and 204 normal weight (94 Black Africans and 110 Mixed Ancestry) adolescents learners. The learners were genotyped for nine polymorphisms (LEP: 19G>A, Lys36Arg, Val94Met; LEPR: Lys109Arg; Gln223Arg, Lys656Asn; CART: c.160-33G>A, c.499delA, and c.517A>G; GHRL: Leu72Met; and MC3R: Thr6Lys, Val81Ile) using allele-specific restriction enzyme analysis and automated sequencing. Genotype and haplotype associations with anthropometric variables such as body mass index (BMI), waist, hip, and mid-upper-arm circumferences (WC, HC, MUAC), and metabolic traits (fasting blood glucose, high density lipoproteincholesterol, total cholesterol), and blood pressure were further conducted. Furthermore, the type and frequency of physical activity was assessed by means of structured questionnaires; and its effect on obesity-related variables investigated in learners that were genotyped for the MC3R Thr6Lys and Val81Ile polymorphisms. Results: In a stepwise backward logistic regression analysis (containing age, gender, and LEP, LEPR, CART and GHRL polymorphisms), CART c.517A>G was independently significantly associated with obesity (OR= 5.98; 95%CI= 2.02, 21.27). CART c.517G carriers had higher MUAC (b coefficient= 1.88; 95%CI= 0.31, 3.44) while the LEPR 109Arg allele was significantly associated with decreased BMI (b coefficient = -2.36; 95%CI= -4.24, -0.47), WC (b coefficient = -5.66; 95%CI= -9.89, -1.44) and MUAC (b coefficient = -1.61; 95%CI= -3.00, -0.22); after adjusting for age, gender, and ethnicity. The haplotype containing the three LEP polymorphisms (A-A-A compared to the reference G-A-G haplotype) increased BMI (p= 0.0155), MUAC (p= 0.0146), and HC (p= 0.0128). The minor alleles of the MC3R polymorphisms decreased BMI, HC, WC, MUAC and TC; whilst only the Thr6Lys was associated with systolic and diastolic blood pressure (p= 0.0047 and 0.0027, respectively) in Mixed Ancestry learners. Doing house chores was associated with lower total cholesterol, independently and in the presence of the 81Ile allele (b coefficient = -0.355; 95%CI= 0.148, 0.561). Conclusion: To our knowledge, this is the first study that reports CART c.517A>G polymorphism as a risk factor for obesity in adolescents. Furthermore, the present study demonstrated that the MC3R polymorphisms had a positive effect on total cholesterol, which was further enhanced in physically active individuals. Similar to other studies, LEPR Lys109Arg and LEP polymorphisms were associated with variations in obesity-related variables among Black African and Mixed Ancestry South African learners.
AFRIKAANSE OPSOMMING: Agtergrond/Doelwitte: Vetsug het drasties toegeneem in Suid-Afrikaanse kinders en adelossente met ‘n beduidende variasie opgemerk tussen verskillende rassegroepe. Gene verantwoordelik vir regulering van eetlus is reeds wêreldwyd in verskillende bevolkingsgroepe bestudeer, maar hul rol in oorgewig Suid-Afrikaanse adolessente is onbekend. Die huidige studie was daarop gerig om ondersoek in te stel na die rol van hierdie gene en hul gekombineerde effek met fisiese aktiwiteit in die ontwikkeling van vetsug onder Suid-Afrikaanse adolessente. Metodes: ‘n Totaal van 1564 Suid-Afrikaanse leerders van Kaukasiese Afkoms (n=146), Gemengde Afkoms (n=872) en Swart Afkoms (n= 537) was gewerf in die navorsingsprojek wat ten doel gehad het om kinders en adolosente met diabetes en die metaboliese sindroom te identifiseer wat skole bygewoon het in semi-voorstedelike gebiede van die Wes-Kaap. Die huidige gevalle studie het 227 vetsugtige-oorgewig (115 Swart Afkoms en 110 Gemengde Afkoms) en 204 normale gewig (94 Swart Afkoms en 110 Gemengde Afkoms) leerders ingesluit. Die leerders was gegenotipeer vir nege polimorfismes (LEP: 19G>A, Lys36Arg, Val94Met; LEPR: Lys109Arg; Gln223Arg, Lys656Asn; CART: c.160-33G>A, c.499delA, and c.517A>G; GHRL: Leu72Met; and MC3R: Thr6Lys, Val81Ile) met die gebruik van alleel-spesifieke restriksie ensiem analises en geoutomatiseerde DNA volgorde bepalings tegnieke. Genotipiese en haplotipiese assosiasies met antropometriese veranderlikes soos liggaamsmassa indeks (BMI), middel-, heup- en mid-boarm omtrek (WC, HC, MUAC), metaboliese tendense (vastende bloed glukose, hoë-digtheid lipoproteïen-cholesterol, totale cholesterol) en bloeddruk was ook uitgevoer. Die tipe en frekwensie fisiese aktiwiteit was geassesseer deur middel van gestruktureerde vraelyste; en die uitwerking daarvan op vetsugverwante veranderlikes ondersoek in leerders wat vir die MC3R Thr6Lys en Val81Ile polimorfismes gegenotipeer was. Resultate: Statistiese ontleding (‘‘stepwise backward logistic regression analysis”), wat ouderdom, geslag en polimorfismes (LEP, LEPR, CART GHRL) ingesluit het, het getoon dat CART c.517A>G betekenisvol onafhanklik geassosiasieer was met vetsug (OR= 5.98; 95% CI= 2.02, 21.27). CART c.517G draers het ‘n hoër MUAC waarde gehad (b koeffisient = 1.88; 95%CI= 0.31, 3.44), terwyl die LEPR 109Arg alleel betekenisvol geassosieer was met verlaagde BMI ((b koeffisient = -2.36; 95%CI= -4.24, -0.47), WC (b koeffisient = -5.66; 95%CI= -9.89, -1.44) en MUAC (b koeffisient = -1.61; 95%CI= -3.00, -0.22) na die aanpassing van ouderdom, geslag en etnisiteit. Die haplotipe met die drie LEP polimorfismes (A-A-A teenoor die G-A-G verwysingshaplotipe) het die BMI (p= 0.0155), MUAC (p= 0.0146) en HC (p= 0.0128) verhoog. Die mindere allele van die MC3R polimorfismes het die BMI, HC, WC, MUAC en TC verlaag; terwyl slegs die Thr6Lys polymorfisme met sistolies en diastolies bloeddruk (p= 0.0047 en p= 0.0027, onderskeidelik) geassosieer was in Gemengde Afkoms leerders. Die verrigting van algemene huistake was geassosieer met laer totale kolesterol vlakke, onafhanklik en in die teenwoordigheid van die 81lle alleel (b koeffisient= -0.355; 95%CI= 0.148, 0.561). Gevolgtrekking: Na ons wete is hierdie die eerste studie wat die CART c.517A>G polimorfisme as ‘n risikofaktor vir vetsug in adolessente aantoon. Die huidige studie toon ook dat die MC3R polimorfisme ‘n positiewe effek op totale kolesterol gehad het, wat ook verder versterk was in fisiese aktiewe individue. Soortgelyk aan ander studies, was die LEPR Lys109Arg en LEP polimorfismes geassosieer met variasies in vetsug-verwante veranderlikes onder Suid-Afrikaanse Swart en Gemengde Afkoms leerders.
This research was supported by a grant from the University Research Fund of the Cape Peninsula University of Technology, Harry Crossley, University of Stellenbosch, Faculty of Health Sciences, Medical Research Council, and the National Health Laboratory Services, South Africa.

Thesis (MPath)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Major Depressive Disorder (MDD) is a severe debilitating medical condition that may lead to suicide. Due to a poor understanding of the biological mechanisms underlying the disease process therapeutic decisions are usually taken using a ‘trial and error’ approach. This is not ideal since many treatments do not work as expected for all individuals. Studies have shown that only half of MDD patients receive the appropriate treatment, whereas many patients have adverse response to anti-depressants. These may include weight gain and raised homocysteine levels that may further compromise the health status of MDD patients and may partly explain the link with cardiovascular disease. The objective of the study was to identify genetic risk factors interacting with environmental factors implicated in MDD that may be of relevance to the South African population. Polymorphisms in the MTHFR (677 C>T, rs1801133 and 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) and SLC6A4 (43 bp ins/del, rs4795541) genes were genotyped in 86 MDD patients and 97 population-matched controls. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MDD. A total of 183 unrelated Caucasians participated in the study, including 69 females and 17 males with MDD and 57 female and 40 male controls without a personal and family medical history of overlapping stress/anxiety and depressive disorders. All study participants were genotyped for the six selected SNPs considered clinically useful based on international data. The allelic distribution of the SNPs, single or combined into a genotype risk score after counting their minor alleles, did not differ between MDD patients and controls. Homocysteine levels were determined and correlated with body mass index (BMI) and other variables known to influence these phenotypes. The folate score assessed with use of the study questionnaire was significantly lower in the patient group compared with controls (p=0.003) and correlated significantly with BMI, particularly in females (p=0.009). BMI was on average 8% higher in the MDD patients compared with controls (p=0.015) after adjustment for age and sex. The MTHFR rs1801133 677 T-allele was associated with a 14% increase in BMI in MDD patients but not controls (p=0.032), which in turn was associated with significantly increased homocysteine levels (p<0.05). The aims of the study were successfully achieved. Identification of the MTHFR rs1801133 677 T-allele reinforces the importance of adequate folate intake in the diet due to increased risk of obesity and depression found to be associated with low dietary intake. Evidence of shared genetic vulnerability for many chronic diseases and drug response mediated by the MTHFR 677 T-allele support the clinical relevance of this low-penetrance mutation.
AFRIKAANSE OPSOMMING: Major depressie (MD) is ‘n aftakelende siektetoestand wat tot selfdood kan lei. Onkunde oor die siekte se onderliggende biologiese meganismes lei dikwels tot ‘n lukrake terapeutiese benadering. Dit is ‘n onbevredigende situasie aangesien indiwidue verskillend reageer op die middels wat voorgeskryf word. Navorsing toon dat slegs ongeveer die helfte van MD pasiënte toepaslike behandeling kry, terwyl anti-depressante ‘n nadelige uitwerking het op baie pasiënte. Dit sluit massatoename en verhoogde homosisteïenvlakke in wat MD pasiënte se gesondheid bykomend nadelig kan beïnvloed en die verband met kardiovaskulêre siekte gedeeltelik kan verklaar. Hierdie studie poog om MD verwante genetiese risikofaktore en omgewingsfaktore wat mekaar beïnvloed en moontlik op die Suid Afrikaanse bevolking betrekking het, te identifiseer. Polimorfismes in die MTHFR (677 C>T, rs1801133 en 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) en SLC6A4 (43 bp ins/del, rs4795541) gene is geanaliseer in 86 MD pasiënte en 97 kontroles geselekteer van dieselfde populasie. Die spesifieke doelwitte was om 1) hoë deurset direkte polimerase kettingreaksie (RT-PCR) genotiperingstoetse vir die 6 gekose polimorfismes met direkte volgordebepaling as maatstaf analities te valideer vir 2) moontlike insluiting in ‘n patologie-ondersteunde genetiese toetsstrategie met die oog op beter kliniese hantering van MD. Altesaam 183 Kaukasiërs het aan die studie deelgeneem. Die MD pasiënte het uit 69 vroue en 17 mans bestaan. Die kontroles (57 vroue en 40 mans) het geen mediese geskiedenis (persoonlik of familie) van oorvleuelende stress/angstigheid of depressie gehad nie. Gebaseer op internasionale data, is al die deelnemers vir die 6 gekose, potensieel klinies-bruikbare polimorfismes getoets. Die alleliese verspreiding van die polimorfismes enkel of gekombineer (uitgedruk as ‘n genotipe-risiko-syfer nadat minor allele getel is), was dieselfde in MD-pasiënte en kontroles. Homosisteïenvlakke is bepaal en gekorreleer met die liggaamsmassa-indeks (BMI) en ander veranderlikes wat bekend is vir hulle invloed op hierdie fenotipes. In teenstelling met die kontroles, was die folaat telling, soos bepaal met die studievraelys, betekenisvol laer in die pasiënte (p=0.003). Die korrelasie met die liggaamsmassa-indeks, spesifiek by vroue, was ook betekenisvol (p=0.009). Na aanpassings vir ouderdom en geslag, is gevind dat die liggaamsmassa-indeks gemiddeld 8% hoër was in die die MD pasiënte teenoor die kontroles. By MD-pasiënte, maar nie by die kontroles nie, is die MTHFR rs1801133 677 T-alleel geassosieer met ‘n 14% toename in liggaamsmassa-indeks (p=0.032), wat ook geassosieer was met betekenisvolle verhoogde homosisteïenvlakke (p<0.05). Die doelwitte van die studie is bereik. Identifisering van die MTHFR rs1801133 677 T-alleel beklemtoon hoe belangrik dit is om voldoende folaat in te neem, veral omdat ‘n verhoogde risiko vir vetsug en depressie met ‘n lae folaatinname in die diet geassosieer word. Die kliniese belang van die MTHFR 677 T-alleel word beklemtoon deur toenemende bewyse wat daarop dui dat gedeelde genetiese vatbaarheid vir ‘n verskeidenheid van kroniese siektes asook middelrespons aan bemiddeling deur hierdie lae penetrasie mutasie toegeskryf kan word.
Winetech
Technology for Human Resources and Industry Program (THRIP).

Thesis (MSCMedSc)--Stellenbosch University, 2012.
Includes bibliography
ENGLISH ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world. The disease spectrum of NAFLD extends from steatosis (types 1,2) to non-alcoholic steatohepatitis (NASH) with inflammation (types 3,4). The aims of the study were 1) to analytically validate high-throughput real time polymerase chain reaction (RT-PCR) assays for three selected single nucleotide polymorphisms (SNPs), FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) and PPARγ rs1801282 (Pro12Ala, 34 C>G), and 2) to perform genotype-phenotype association studies in relation to biochemical abnormalities, disease severity and age of onset. A total of 119 patients with fatty liver identified on ultrasound, including 88 histologically confirmed NAFLD patients, and 166 control individuals were genotyped for the three selected SNPs. RT-PCR validated against direct sequencing as the gold standard was used for detection of genetic variation. All three SNPs were in Hardy Weinberg equilibrium in the study population, except for a deviation in genotype distribution detected for PPARγ rs1801282 in the NAFLD patient subgroup (p<0.001). After adjustment for age and gender, the risk-associated FTO rs9939609 A-allele was detected at a significantly higher frequency in the Caucasian compared with Coloured patients (p=0.005). The opposite was detected for the risk-associated TNF-α rs1800629 A-allele, which occurred at a significantly higher frequency in the Coloured compared with Caucasian NAFLD patients (p=0.034). The onset of fatty liver disease symptoms was on average 5 years younger in the presence of each risk-associated TNF-α rs1800629 A-allele (p=0.028). When considered in the context of an inferred genotype risk score ranging from 0-6, disease onset occurred on average 3 years earlier (p=0.008) in the presence of each risk-associated FTO A-allele, TNF-α A-allele or PPARγ C-allele. After adjustment for age, gender and race, no differences in genotype distribution or allele frequencies were observed between histologically confirmed NAFLD (types 1,2) and NASH (types 3,4) patients, while the minor allele frequency for the TNF-α rs1800629 was significantly higher in the total NAFLD (types 1-4) (p=0.047) as well as NASH subgroup (NAFLD types 3,4) (p=0.030) compared with obese patients without a histologically confirmed NAFLD diagnosis. A significant correlation was furthermore observed between the number of TNF-α rs1800629 A-alleles and increasing CRP levels (p=0.029), with a favourable reduced effect in the presence of low- to moderate alcohol intake. The average waist circumference of physically active NAFLD patients was 12% lower than in physically inactive patients (p=0.004). In view of the results presented in this study, the inclusion of the selected SNPs, and in particular the pro-inflammatory TNF-α rs1800629 polymorphism, may be considered as part of a comprehensive cardiovascular risk evaluation of NAFLD patients. Ultimately, early detection of patients with fatty liver disease symptoms and effective intervention based on the underlying disease mechanism may prevent progression from NAFLD to NASH, shown to be an independent risk factor for cardiovascular diseases.
AFRIKAANSE OPSOMMING: Nie-alkoholiese lewervervetting (NALV) is die mees algemene kroniese lewersiekte in die wêreld. Die siektespektrum van NALV strek van steatose (vervette lewer tipes 1,2) tot steatohepatitis met inflammasie (NASH tipes 3,4). Die doel van die studie was 1) om analities die hoë omset polimerase kettingreaksie (RT-PKR) metode te valideer vir die geselekteerde enkel nukleotied polimorfismes (ENPs) FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) en PPARγ rs1801282 (Pro12Ala, 34 C>G), en 2) om genotipe-fenotipe assosiasie studies uit te voer ten opsigte van relevante biochemiese abnormaliteite, graad van die siekte en aanvangsouderdom. ’n Totaal van 119 pasiënte met vervette lewers is geïdentifiseer met behulp van ultraklank, insluited 88 histologies-bevestigde NALV pasiënte, en 166 kontrole individue. Hierdie pasiënte is gegenotipeer vir die 3 geselekteerde ENP’s. RT-PKR gevalideer met direkte DNA volgorde bepaling as die goue standaard, is gebruik vir opsporing van genetiese variasie. Al die ENP’s was in Hardy Weinberg ekwilibrium in die studie populasie, behalwe vir ’n afwyking in genotipe verspreiding waargeneem vir PPARγ in die NALV subgroep (p<0.001). Nadat aanpassings gemaak is vir ouderdom en geslag, is die risiko-geassosieerde FTO rs9939609 A-alleel waargeneem teen ’n betekenisvol hoër frekwensie in die Kaukasiese pasiënte in vergelyking met Kleurling pasiënte (p=0.005). Die teenoorgestelde is waargeneem vir die risiko-geassosieerde TNF-α rs1800629 A-alleel wat voorgekom het teen ’n betekenisvol hoër frekwensie in die Kleurling NALV pasiënte, in vergelyking met Kaukasiese NALV pasiënte (p=0.034). Die aanvang van NALV was gemiddeld 5 jaar vroeër in die teenwoordigheid van elke risiko-geassosieerde TNF-α rs1800629 A-alleel (p=0.028). Met inagneming van ’n genotipe risiko telling tussen 0–6, het aanvang van siekte gemiddeld 3 jaar vroeër voorgekom (p=0.008) in die teenwoordigheid van elke toenemende risiko-geassosieerde FTO A-alleel, TNF-α A-alleel en PPARγ C-alleel. Nadat aanpassings gemaak is vir ouderdom, geslag en ras, is geen verskille waargeneem in genotipe verspreiding of alleel frekwensies tussen histologies bevestigde NALV (tipes 1,2) en NASH (tipes 3,4) pasiënte nie, terwyl die minor alleel telling vir die TNF-α rs1800629 betekenisvol hoër was in die totale NALV (tipes 1–4) (p=0.047) asook die NASH subgroep (NALV tipes 3,4) (p=0.03) in vergelyking met vetsugtige pasiënte sonder ’n histologies bevestigde diagnose. ‘n Statisties beteknisvolle korrelasie is verder waargeneem tussen die aantal TNF-α rs1800629 A-allele en toenemende CRP vlakke (p=0.029), met n gunstige verlaagde effek in die teenwoordigheid van lae alcohol gebruik. Die gemiddelde middellyf-omtrek van fisies aktiewe NALV pasiënte was 12% minder as fisies onaktiewe pasiente (p=0.004). Na aanleiding van die resultate van hierdie studie behoort insluiting van geselekteerde ENP’s, en in besonder die pro-inflammatoriese TNF-α rs1800629 polimorfisme, as deel van ’n omvattende kardiovaskulere risiko evaluasie oorweeg te word. Aan die einde van die dag mag vroeë identifikasie van NALV pasiente en effektieve intervensie gebasseer op die onderliggende siekte meganisme, vordering tot NASH verhoed wat getoon is om ’n onafhanklike risiko faktor vir kardiovaskulêre siekte te wees.
Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011.
Includes bibliography.
ENGLISH ABSTRACT: Genetic screening to improve obesity treatment outcomes is available despite the lack of conclusive evidence, specifically for Caucasian South Africans, in this regard. The aim of this study was to investigate the association between genotype (seven polymorphisms) and body mass index (BMI), health and lifestyle indicators in a cross-sectional sample of overweight/obese Caucasian adults (n=133), as well as the association between genotype and weight loss outcomes following an intervention (n=88) using a quasi experimental study design (time-series). The intervention consisted of a 24-week conservative weight loss programme that included dietary, physical activity and behavioural components. The primary null hypothesis for the cross-sectional sample, namely that there is no association between genotype and BMI, has not been rejected. A number of the secondary/exploratory hypotheses were rejected of which the most plausible associations (based on support by the literature and a physiological basis for the findng) are: 1) the mutant TT homozygotes of the GNB3 C825T polymorphism may have a higher risk to develop the metabolic syndrome (MetS) as they had significantly higher fasting triglyceride and glucose levels, a higher number of traits that met the diagnostic cut-off criteria for MetS and higher number of these subjects was diagnosed with MetS compared to the wild-type C-allele carriers; and 2) subjects with mutant alleles of either the FTO rs1421085 or rs17817449 polymorphisms may have poorer eating behaviours (a higher rigid control, habitual and emotional disinhibition, perceived hunger and internal locus for hunger) and higher intake of high-fat foods. The primary null hypothesis for the intervention sample, namely that there is no association between genotype and weight loss outcome, was not rejected for the FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg and GNB3 C825T polymorphisms. However, it was rejected in some instances indicating the following associations: 1) The wild-type TT homozygotes of the FTO rs17817449 polymorphism lost significantly more weight during the first two months of the program compared to the mutant allele carriers (this is a novel finding); 2) The wild-type Arg16Arg homozygotes of the ADRB2 Arg16Gly polymorphism lost significantly more weight during the first month of the program compared to the mutant allele carriers (this finding is supported by one other intervention study); 3) Subjects with a mutant C-allele of the INSIG2 rs7566605 polymorphism and a mutant Gly16-allele of the ADRB2 Arg16Gly polymorphism lost significantly less weight over the six month intervention period (this is a novel genegene interaction finding). A number of secondary/exploratory hypotheses were rejected, of which the most plausible finding include that the improvement in emotional disinhibition in the wild-type TT subjects of the FTO rs1421085 polymorphism was associated with a more pronounced decrease in BMI over the six month weight loss period. The integration of the results from this study with the literature indicates that there is insufficient evidence at this stage for genetic screening of the polymorphisms investigated in this study and the provision of evidence-based personalized recommendations for weight loss in obese individuals. It is recommended that these associations should be viewed as priority in future research.
AFRIKAANSE OPSOMMING: Genetiese sifting om die resultate van vetsug behandeling te verbeter is beskikbaar ten spyte van ‘n tekort aan genoegsame bewyse, spesifiek ten opsigte van Kaukasiërs van Suid-Afrika. Die doel van hierdie studie was om die assosiasie tussen genotipe (sewe polimorfismes) en liggaamsmassa indeks (LMI), gesondheid en lewenstyl indikatore in ‘n dwarssnit (cross-sectional) steekproef van oorgewig/vetsugtige Kaukasiër volwassenes (n=133) te ondersoek, asook die assosiasie tussen genotipe en gewigsverlies uitkomste na afloop van ‘n intervensie (n=88) in ‘n kwasi-eksperimentele studie ontwerp (tydreeks). Die intervensie het bestaan uit ‘n 24-week konserwatiewe gewigsverlies program met dieet, fisieke aktiwiteit en gedragskomponente. Die primêre nul hipotese vir die dwarsnit steekproef, naamlik dat daar geen assosiasie tussen genotipe en LMI is nie, is nie verwerp nie. ‘n Aantal sekondêre/spekulatiewe hipotesis is verwerp waarvan die mees geloofwaardige assosiasies (gebasseer op ondersteuning van die literatuur en ‘n fisiologiese basis vir die bevinding) die volgende insluit: 1) die mutante TT homosigote van die GNB3 C825T polimorfisme het moontlik ‘n hoër risiko vir die ontwikkeling van die metaboliese sindroom (MetS) aangesien hulle betekenisvolle hoër vastende trigliseriede en glukose vlakke gehad het, ‘n grooter aantal kenmerke gehad het wat aan die diagnostiese afsnykriteria vir MetS voldoen en ‘n grooter aantal van hierdie persone was met MetS gediagnoseer in vergelyking met die wilde-tipe C-alleel draers; en 2) persone met die mutante allele van die FTO rs1421085 of rs17817449 polimorfismes het moontlik ‘n swakker eetgedrag (‘n hoër rigiede kontrole, gewoonte en emosionele disinhibisie, waarneembare honger en interne lokus van honger) en ‘n hoër inname van hoë-vet voedsel. Die primêre nul hipotese vir die intervensie steekproef, naamlik dat daar geen assosiasie tussen genotipe en gewigsverlies uitkomste is nie, is nie vir die FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg en GNB3 C825T polimorfismes verwerp nie. Dit was egter in sommige gevalle vir die volgende assosiasies verwerp: 1) Die wilde-tipe TT homosigote van die FTO rs17817449 polimorfisme het betekenisvol meer gewig in die eerste twee maande van die program verloor in vergelyking met die mutante alleel draers (dit is ‘n nuwe bevinding); 2) Die wilde-tipe Arg16Arg homosigote van die ADRB2 Arg16Gly polimorfisme het betekenisvol meer gewig gedurende die eerste maand van die program verloor in vergelyking met die mutante alleel draers (hierdie bevinding word ondersteun deur een ander intervensie studie); 3) Persone met ‘n mutante C-alleel van die INSIG2 rs7566605 polimorfisme en ‘n mutante Gly16-allele van die ADRB2 Arg16Gly polimorfisme het minder gewig tydens die ses maande intervensie periode verloor (dit is ‘n nuwe geen-geen interaksie bevinding). ‘n Aantal sekondêre/ spekulatiewe hipoteses is verwerp, waarvan die mees geloofwaardigste bevinding insluit dat ‘n verbetering in emosionele disinhibisie van die wild-tipe TT persone van die FTO rs1421085 polimorfisme geassosieer was met ‘n meer prominente daling in LMI oor die ses maande gewigsverlies periode. Die integrasie van die resultate van hierdie navorsing met die literatuur dui aan dat daar op hierdie stadium onvoldoende bewyse vir genetiese sifting en die voorsiening van bewys-gebasseerde persoonlike aanbevelings vir gewigsverlies in vetsugtig individue bestaan vir die polimorfismes wat ondersoek is. Dit word aanbeveel dat hierdie assosiasies as prioriteit in toekomstige navorsing beskou moet word.

Obesity, or the presence of an excessive amount of body fat is a major public health problem in the United States and, increasingly, the rest of the world. The apparent drivers of the increased prevalence of obesity over the past several decades are environmental changes, e.g., dietary and lifestyle changes that interact with the individual’s genetic susceptibility for weight gain. In humans, obesity appears to be driven primarily by increases of energy intake relative to expenditure; that is, to uncompensated hyperphagia. The heritability of adiposity, i.e., the extent to which differences in adiposity among individuals living in the same environment can be attributed to genetic differences is estimated by twin and other studies to be about 50%. Large scale population-based association studies (e.g., GWAS) have suggested that genetic variants (e.g., SNPs) associated with susceptibility or resistance to obesity affect primarily the development and regulation of the central nervous system (CNS). In particular, SNPs in genes that play a role in brain cellular structures and molecular pathways known to regulate energy homeostasis, most notably, the leptin-melanocortin signaling pathway, are among the most highly associated with human obesity. For example, SNPs around the melanocortin receptor, MC4R, are associated with increased adiposity and mutations in MC4R represent the most prevalent genetic variations associated with monogenic obesity. Ultimately, however, relatively little is understood about the biological mechanisms by which an individual’s genetic sequence confers susceptibility or resistance to weight gain in a specific environment. Such understanding could open new avenues for the prevention and treatment of obesity and would advance our understating of genetic predisposition to other complex diseases. The goal of this research is to identify genomic regions contributing to susceptibility and resistance to hyperphagic obesity by analysis of whole genome sequence and hypothalamic gene expression data from two genetically related cohorts of Sprague-Dawley rats – the ‘Levin Rat’. Dr. Levin developed these animals by successive generations of selective breeding for differences in adiposity resulting from exposure to a calorically dense, highly palatable diet (described in detail in Chapter 2). These selectively bred diet-induced obese (DIO) and diet-resistant (DR) Levin rats have been the topic of a large body of physiological research (reviewed in Chapter 1) showing potentially important similarities to the physiology of human obesity. In particular, implication of diet-sensitive hyperphagia as the primary driver for the differential susceptibility of DIO (diet-induced obese) animals to gain weight in response to palatable diet; neuroanatomical and functional differences between DIO and DR in hypothalamic nuclei (e.g., ARH, PVH) and leptin signaling, prior to the development of obesity; and, neurophysiological differences between DIO and DR (diet-resistant) in ‘reward circuit’ nuclei (e.g., NAc) and their differential responses to pharmacological stimuli, e.g., cocaine, as well as palatable diet. These findings established the Levin rat as an interesting model for aspects of the biology of human obesity. Importantly, the genetic bases for these Levin rat phenotypes have remained unknown. Our efforts to elucidate the underlying genetics of this model system are, therefore, of potential relevance to human obesity. We obtained phenotypic, whole genome sequence (WGS) and hypothalamic gene expression (RNA-Seq) data from selected Levin rats and analyzed these data to identify several loci that are highly associated with the body weight phenotype in the Levin cohorts, as well as in a confirmation cohort of genetically related progeny being studied for phenotypes related to addictive behaviors. In Chapter 2, I describe our methods and approaches to collecting the relevant phenotypic and genetic data, and to selecting primary and confirmation cohorts for the WGS and RNA-Seq studies. In Chapter 3, I describe our bioanalytical and statistical approaches to the WGS data designed to detect genomic loci likely inherited Identical by Descent (IBD) from common ancestors of the DIO, or DR animals; such loci constitute candidate obesity susceptibility loci for the Levin rat. In Chapter 4, I present the phenotypic differences between the DIO and DR animals used for the study, the results of IBD analysis of the WGS, and indicate genes we found to be differentially expressed or spliced in their respective hypothalami. I also show, using confirmation groups (CG) of animals from the primary cohorts of Levin rats (not used for IBD mapping), that the identified susceptibility loci are highly associated with DIO/DR lineage. In Chapter 5 I show, using representative SNPs from the loci with the highest association to the obesity susceptibility phenotype, significant association between the ‘DIO’ genotype and increased body weight in an independent cohort of Levin rat progeny [designated Obesity Prone (OP) and Obesity Resistant (OR)] maintained by Dr. Ferrario (U. Michigan). Using gene set enrichment analyses (GSEA), I show that the candidate susceptibility loci are enriched in CNS genes and genes previously associated with obesity traits in human GWAS. I also analyzed the genes implicated by differential expression and/or splicing (RNA-Seq) in relation to the genetic susceptibility map (IBD analyses) and identified several genes whose hypothalamic differential regulation (e.g., expression, splicing) may be linked to genetic sequence variations between the DIO and DR animals. Interestingly, essentially, all of the identified genes have been previously implicated in body weight regulation in other species, including humans. Finally, I propose future studies to build upon this work in order to further refine the genetic susceptibility map, test the roles of putative candidate genes, and ultimately elucidate the genetic bases for the differences in body weight in this genetically complex mammalian model of diet-sensitive obesity: the Levin DIO/DR rats. In summary, the conclusions from these studies are that: - The 15 susceptibility loci we identified, span in total ~35 Mb, or 1.15% of the rat genome, likely contain a significant portion of the causal alleles underlying the Levin rat phenotype. - The majority of genetic variants in these susceptibility loci are ‘non-coding,’ e.g., intronic, in untranslated regions, or intergenic – similar to common obesity SNPs. - Our susceptibility map is enriched for genes governing CNS function and development, and its human syntenic genes/loci are enriched in obesity SNPs, identified by GWAS. Therefore, Levin rat obesity risk may have a genetic architecture similar to that in humans. - We identified several genes that are differentially expressed or spliced in the hypothalamus of DIO/DR animals, and are also implicated by our genetic map: Zfr, Slc24a2, Fhit, Adarb1, Lrp2. Interestingly, human orthologs of all of these genes have been implicated in obesity by GWAS or familial linkage studies, enhancing them as putative candidates for a role in the Levin rat obesity phenotypes. - Our studies further establish the Levin rat as a model system for polygenic human obesity and lay the foundation for further studies to elucidate the genetic basis of this interesting and important complex trait of hyperphagic diet-sensitive obesity.

Background: The obesity epidemic is the greatest public health problem of our time, and exerts an enormous health and economic burden by acting as a risk factor for multiple disorders and all-cause mortality. While environmental and social factors certainly contribute to the complex etiology of obesity, there is strong evidence of a substantial genetic component. The majority of obesity genes are involved the leptin-melanocortin receptor pathway governing energy homeostasis, but mutations affecting this circuit are often untreatable and rare, and an improved understanding of other genetic risk factors could aid in the development of novel therapies. In this thesis I study two obesity candidate genes with unclear direct relevance to disease: 1) rare structural variation at the 16p11.2 BP4-BP5 locus and 2) common variation in the Fat Mass and Obesity-Associated (FTO) gene. Methods: 1) I analyzed disinhibited eating measurements from families with 16p11.2 copy number variation (CNV) carriers, to test whether eating in the absence of hunger (EAH) and loss of control (LOC) eating behaviors mediate the dosage-dependent CNV-BMI relationship. 2) Using association data from a study of over 20,000 African Americans and 1,145 functional annotations from the Encyclopedia of Non-coding Elements (ENCODE) and Roadmap Epigenomics projects, I statistically fine-mapped the FTO locus to identify the SNP(s) and cellular contexts underlying the association between FTO and obesity. Results: 1) EAH due to external triggers mediates over 30% of the 16p11.2 deletion’s effect on obesity, while other EAH and LOC behaviors were not significant mediators. This result was independent of IQ deficits and autism related to the CNV, as well as parents’ feeding behaviors and practices. 2) Given 51 FTO SNPs’ association statistics, correlation, and overlap with functional annotations, rs9927317 and rs62033405 had the highest posterior probability of association with obesity. Obesity-associated SNPs may regulate expression of FTO and/or nearby genes through the activity of enhancers and 5’ ends of transcribed genes in the substantia nigra of the brain, bone chondrocytes, and white adipose. Conclusions: These results may help pinpoint the specific genes, regulatory elements, and cellular contexts through which the 16p11.2 and FTO loci exert their effects on obesity.

The primary goal was to study the influence of adipocyte number and volume, inflammation, insulin resistance, and genetic factors on indicators of liver injury, surrogate marker of non alcoholic fatty liver disease (NAFLD). The secondary goal was to explore the occurrence of NAFLD and its relationship with variations in liver function biomarkers. The first objective was to determine the association of plasma levels of monocyte chemoattractant protein-1 (MCP-1) with omental adipocyte number, insulin resistance and circulating concentrations of liver injury markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in unrelated baboons. Significant associations of MCP-1 with other measured traits were established. The second objective was to examine if adiposity-related parameters are under genetic influence and to evaluate their genetic correlations with AST in pedigreed baboons. Adipocyte volume and number, body weight and plasma AST were heritable. Genetic correlations between adiposity-related phenotypes and AST were significant. A genome wide scan yielded a strong signal for adipocyte volume on chromosome 6. The third aim was to explore the genetic factors that influence variations in plasma levels of [gamma] glutamyl transferase (GGT) and albumin (ALB), and to evaluate their genetic correlations with cardiovascular risk factors in pedigreed baboons. Significant linkages for GGT and albumin were identified on chromosome 20_22 and chromosome 10, respectively. Genetic correlations between ALB and cardiovascular risk factors were significant. No statistically significant associations were found between GGT and cardiovascular-related phenotypes. The fourth objective was to investigate the prevalence of NAFLD and its association with altered liver protein levels in unrelated baboons. The influence of weight and insulin resistance on the occurrence of NAFLD was inconclusive. Significant relationships between the variations in plasma levels of liver injury biomarkers and severity of the disease could not be established. In conclusion, the first three studies provided observational and genetic evidence of a relationship between liver function markers and adiposity-related factors in baboons. However, the results of the fourth study do not provide conclusive evidence to suggest that body weight and insulin resistance play a significant role in the development of NAFLD in these baboons.

The accumulation of excessive fat is a serious concern in both the livestock production and human health fields. Obesity is a condition of excessive energy storage in the form of body fat ( triacylglycerols ). The cellular basis for obesity is not yet understood but numerous factors have been suggested. Genetic factors and altered metabolism may be two cellular parameters that contribute to the excessive accumulation of fat. Adipocytes are responsive to extracellular signals, which have a dramatic effect on their metabolism implying that these metabolic responses may be the result of differences in the composition or responsiveness of adipocyte receptors. The purpose of this research was to identify adipocyte specific marker proteins and to determine if there are any differences in the expression of these proteins that may be associated with the conditions of genetic obesity or leanness. Identification of adipocyte-specific markers should allow for a better understanding of adipocyte growth and development and determination of the adipocytes role in energy metabolism. A hybridoma line was produced which secreted a monoclonal antibody (LA-1) directed against a novel 64-kD protein unique to porcine adipocyte plasma membranes, having an undetermined function in the unique physiology of the adipocyte. This protein was found to be expressed in genetically lean adipocytes but not adipocytes derived from genetically obese sources. In order to elucidate the role of this unique adipocyte-specific plasma membrane protein, a porcine adipocyte eDNA library was produced. This library was screened with LA-1 and a eDNA clone isolated. This eDNA clone was used to study the expression of the gene responsible for this unique protein at the nucleic acid level. Northern blot analysis revealed a 5000- and a 7000-base pair species of poly (A+) RNA present in total RNA isolated from contemporary porcine adipose tissue. Determination of the nucleic acid sequence of the eDNA clone should allow for the determination of the actual identity and possible function of this adipocyte-specific protein and the possible role it may serve in regulating adipocyte growth and development.
Graduation date: 1991

The foz/foz mouse is a murine model of Alstr{u00F6}m syndrome, a monogenetic disorder characterised in humans by childhood obesity, hearing loss, blindness, hyperinsulinaemia, early-onset type 2 diabetes and liver disease. In 2006, research from the host laboratory reported that foz/foz mice inherit a spontaneous mutation (foz) , an 11 base pair deletion in exon 8 of the Alms1 gene, and develop a similar phenotype to patients suffering from Alstr{u00F6}m syndrome. Thus, foz/foz mice are obese and exhibit high circulating insulin and leptin levels as well as fatty liver disease and metabolic syndrome. The purpose of the studies presented in this thesis was to further characterise the pathogenesis of obesity in foz/foz mice, by studying the role of Alms1 and hypothalamic appetite-regulating neuropeptide expression during the development of obesity. ALMS1 has been shown to localise at the base of primary cilia in what is termed the basal body or centrosome. Primary cilia are ubiquitously expressed hair-like organelles. Therefore, the first approach was to study primary cilia in the hypothalamus as well as hypothalamic Alms1 gene expression and Alms1 localisation in foz/foz and wildtype (WT) mice from birth until the obese phenotype is evident. At birth, foz/foz mice showed similar number of ciliated hypothalamic neurons to WT mice. However, from weaning and onwards the number of cilia was significantly decreased in foz/foz mice. In addition, while cilia were present in primary neuronal cultures from foz/foz and WT mice, Alms1 was only detected in WT neurons, appearing as two perinuclear dots at the base of cilia. After weaning, serum leptin levels become greatly elevated in foz/foz compared to WT mice. Leptin decreases appetite by acting in the hypothalamus and inducing or inhibiting the release of appetite-regulating neuropeptides. A detailed study of key hypothalamic neuropeptides demonstrated no differences in their gene and/or protein expression or localisation between foz/foz and WT mice. This failure of elevated leptin levels to decrease appetite and body weight is defined as leptin resistance. Further studies were therefore performed on hypothalamic leptin receptor (Ob-R) expression and signalling pathways to characterise leptin resistance in foz/foz mice. These results demonstrated induction of two proteins, SOCS3 and PTP1B, which negatively regulate Ob-R signalling and have been implicated in leptin resistance. Taken together, the data presented in this thesis strongly support the proposal that foz/foz mice develop leptin resistance, which correlates molecularly with over-expression of at least two negative feedback regulators of Ob-R. In addition, the post-natal reduction in ciliated hypothalamic neurons in foz/foz mice, in combination with the lack of Alms1 detection are consistent with the proposal that primary cilia stability/maintenance could be impaired as a consequence of the Alms1 mutation. In conclusion, foz/foz mice provide new opportunities for studying the role of Alms1 and neuronal cilia in appetite regulation, particularly with respect to the onset of leptin resistance. A better understanding of Alms1, cilial stability and behavioural responses that underlie obesity could provide clues to novel therapeutic approaches to combat more common forms of obesity.

Single nucleotides in the first intron of FTO convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes (e.g. RPGRIP1L, IRX3) in the vicinity of FTO. This locus affects food intake, the browning of white adipose tissue and risk of type 2 diabetes (independent of its effects on body weight). FTO and RPGRIP1L expression are decreased in fibroblasts and iPSC-derived human neurons of individuals segregating for obesity risk alleles of FTO at rs8050136 and rs1421085. These alleles exhibit decreased binding of isoform p110 of the CUX1 transcription factor. This isoform activates transcription of both FTO and RPGRIP1L. The FTO locus conveys effects on adiposity via hyperphagia, in part, by regulating FTO and RPGRIP1L expression in the hypothalamus. We examined whether FTO and RPGRIP1L also modify adipogenesis and adipose tissue lipid storage. Such effects would influence systemic consequences of the hyperphagia driven by the actions of the genes in the hypothalamus. Given the role in energy homeostasis of genes encoding elements of the primary cilium, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. In confirmation, we find that Rpgrip1l+/− mice are hyperphagic and obese, and display diminished suppression of food intake in response to leptin administration. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO intronic locus. We describe effects of Rpgrip1l in adipocytes which may contribute to the adiposity phenotype observed in these animals, and possibly humans. Loss of Rpgrip1l in 3T3-L1 preadipocytes increased the number of cells capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes (using Adipoq-Cre) did not increase adiposity in mice fed chow or high fat diet. Neither did we observe any effects of Rpgrip1l knockdown in mature 3T3-L1 adipocytes in vitro. Thus, to the extent that Rpgrip1l affects cell-autonomous adipose tissue function, it appears to do so by effects conveyed in preadipocytes, a cell type in which the primary cilium – as a mediator of developmental signals – may have functional importance. We propose that decreased RPGRIP1L expression in preadipocytes in humans segregating for FTO-associated obesity risk alleles increases the potential storage capacity of adipose tissue. Such capacity would influence the metabolic consequences of positive energy balance due to the action of these alleles within the brain. Fto expression is upregulated during adipogenesis in murine and human cells in vitro, and is more highly expressed in isolated mouse adipocytes than in preadipocytes. Here we demonstrate that FTO is required for the maintenance of adipocyte lipid filling and endocrine function in murine 3T3-L1 cells and human adipose tissue-derived stromal cells. RNAseq analysis indicates that this effect on adipocyte programming is conveyed in part by modulation of C/ebpβ- and C/ebpδ-regulated transcription, consistent with reports that Fto acts a transcriptional coactivator. Fto-/- mice have normal fat mass in early life, but spontaneously lose adipose tissue as they age. We propose that Fto is required to maintain adipocyte viability, a function critical to the prevention of ectopic lipid accumulation in obese states. Such accumulation – both total and in specific anatomic regions – has adverse metabolic consequences. In addition to the developmental effects on adiposity mediated by RPGRIP1L, and the effects conveyed on adipocyte function related to FTO, the FTO locus could also impact systemic energy homeostasis by modifying production of humoral signals that are integrated centrally to regulate energy balance. We explored molecular modifiers of adipocyte production of leptin identified by GWAS that may modify obesity risk. The FTO locus was associated with circulating leptin concentration, but this association was abrogated when corrected for BMI, indicating that this locus does not contribute to adiposity by dysregulating leptin production. Our in vitro findings are consistent in this regard, as knockdown of Rpgrip1l and Fto in 3T3-L1 cells did not affect leptin production per adipocyte. These results, however, are not inconsistent with a role for FTO in maintenance of adipocyte viability.

by Choi Ka Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 113-128).
Abstract and appendix in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
Abstract (Chinese version) --- p.iv
Table of Contents --- p.vi
List of Tables --- p.xi
List of Figures --- p.xiv
List of Abbreviations --- p.xv
Chapter CHAPTER ONE: --- BACKGROUND AND LITERATURE REVIEW
Chapter 1.1 --- Coronary Heart Disease: a global health problem --- p.1
Chapter 1.2 --- Risk Factors of Coronary Heart Disease --- p.3
Chapter 1.2.1 --- Age --- p.3
Chapter 1.2.2 --- Gender --- p.4
Chapter 1.2.3 --- Family History of Cardiovascular Disease --- p.5
Chapter 1.2.4 --- Hypercholesterolemia --- p.7
Chapter 1.2.5 --- Unhealthy Dietary Habits --- p.11
Chapter 1.2.6 --- Obesity --- p.14
Chapter 1.2.7 --- Physical Inactivity --- p.20
Chapter 1.3 --- Clustering of Risk Factors --- p.24
Chapter 1.4 --- Risk Factors in Children: Atherosclerosis Begins Early in Life --- p.26
Chapter CHAPTER TWO: --- RESEARCH IN HONG KONG AND PURPOSES OF THIS STUDY
Chapter 2.1 --- Nutrition Transition --- p.27
Chapter 2.2 --- CHD Mortality Trends in Hong Kong --- p.28
Chapter 2.3 --- Serum Total Cholesterol and Obesity in Hong Kong Adults --- p.29
Chapter 2.4 --- "Obesity, Serum Total Cholesterol, Dietary Habits and Physical Activity of Hong Kong Children and Adolescents" --- p.31
Chapter 2.5 --- Study Purpose and Objectives --- p.35
Chapter CHAPTER THREE: --- SURVEY DESIGN
Chapter 3.1 --- Sample Selection --- p.39
Chapter 3.2 --- "Blood Total Blood Cholesterol, Triglyceride and Anthropometric Measurements" --- p.40
Chapter 3.2.1 --- Total Blood Cholesterol and Triglyceride --- p.40
Chapter 3.2.2 --- Anthropometry Measures --- p.42
Chapter 3.3 --- Questionnaire --- p.45
Chapter 3.3.1 --- Questionnaire Design and Pre-testing --- p.45
Chapter 3.3.2 --- General Health and Socio-demographic Questionnaire --- p.47
Chapter 3.3.3 --- Physical Activity Questionnaire --- p.47
Chapter 3.3.4 --- Dietary Questionnaire --- p.48
Chapter 3.4 --- Data Management --- p.49
Chapter 3.5 --- Statistics --- p.49
Chapter 3.6 --- Data Analysis --- p.50
Chapter 3.6.1 --- Blood Total Cholesterol and Triglyceride --- p.50
Chapter 3.6.2 --- Obesity and Fat Distribution --- p.50
Chapter 3.6.3 --- Diet --- p.51
Chapter 3.6.4 --- Physical Activity Patterns --- p.51
Chapter 3.6.5 --- Body Mass Index of Parent and Family History of Diseases --- p.52
Chapter CHAPTER FOUR: --- RESULTS
Chapter 4.1 --- Sample Size and the Characteristics of the Students in the Two Schools --- p.54
Chapter 4.2 --- Gender and Age Distribution --- p.55
Chapter 4.3 --- Blood Total Cholesterol and Triglyceride --- p.56
Chapter 4.4 --- Anthropometry Measures --- p.58
Chapter 4.5 --- Dietary Habits --- p.60
Chapter 4.5.1 --- Dietary Composition of 3-day Dietary Record --- p.60
Chapter 4.5.2 --- Eating Behaviors --- p.65
Chapter 4.6 --- Physical Activity --- p.68
Chapter 4.7 --- Family History of Diseases --- p.70
Chapter 4.8 --- Parents' Anthropometry --- p.71
Chapter 4.9 --- Demographic Data --- p.71
Chapter 4.10 --- Inter-relationships --- p.75
Chapter 4.10.1 --- Blood Total Cholesterol and Triglyceride --- p.75
Chapter a. --- "Blood Total Cholesterol, Triglyceride and Body Fatness" --- p.75
Chapter b. --- "Blood Total Cholesterol, Triglyceride and Diet" --- p.75
Chapter c. --- "Blood Total Cholesterol, Triglyceride and Physical Activity Patterns" --- p.77
Chapter d. --- Blood Total Cholesterol，Triglyceride and Family History of Hypercholesterolemia --- p.78
Chapter e. --- Relative Importance of the Key Factors in Predicting Blood Total Cholesterol levels --- p.79
Chapter 4.10.2 --- Obesity and Body Fatness --- p.79
Chapter a. --- "Obesity, Body Fatness and Physical Activity Patterns" --- p.79
Chapter b. --- "Obesity, Body Fatness and Diets" --- p.82
Chapter c. --- Body Fatness and Genetics --- p.84
Chapter 4.10.3 --- Diet and Physical Activity --- p.86
Chapter 4.10.4 --- "Blood Total Cholesterol, Triglyceride, Obesity and Other Demographic or Economic Characteristics" --- p.87
Chapter 4.11 --- Clustering of Risk Factors among Obese children --- p.87
Chapter CHAPTER FIVE: --- DISCUSSION
Chapter 5.1 --- Implication of Research Findings --- p.89
Chapter 5.2 --- Limitations --- p.108
Chapter CHAPTER SIX: --- CONCLUSIONS AND RECOMMENDATIONS --- p.111
References --- p.113
Appendices
Chapter I --- Questionnaire (English version) --- p.129
Chapter II --- Questionnaire (Chinese version) --- p.139
Chapter III --- Introductory letter (English version) --- p.152
Chapter V --- Introductory letter (Chinese version) --- p.153
Chapter V --- Consent form (English version) --- p.154
Chapter VI --- Consent form (Chinese version) --- p.155
Chapter VII --- Photos of the standard household measures given to children for estimation of portion size (English version) --- p.156
Chapter VIII --- Photos of the standard household measures given to children for estimation of portion size (Chinese version) --- p.157
Chapter IX --- Responses from the children to the food frequency questionnaire --- p.158
Chapter X --- The frequency of the reported food items liked or disliked by the children --- p.160