Study of genetic variants associated with obesity in Portuguese children

Tese de doutoramento em Antropologia, na especialidade de Antropologia Biológica, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra

The prevalence of obesity is a growing problem worldwide. Such scenario urges for additional efforts in both investment on prevention and research relating to the identification of risk factors that may aid early intervention. It is widely accepted that obesity is a complex multifactorial and heterogeneous condition with an important genetic component in the susceptibility risk. Therefore, the identification of associated gene variants could be essential in the design of prevention strategies and management of individuals genetically predisposed to obesity. In 2007, it was identified the first single nucleotide polymorphism (SNP) located in the FTO gene (rs9939609) associated with obesity by genome-wide association studies (GWAS). Until now, more than 52 genetic loci have been unequivocally associated with obesity related-traits in several European populations. However, none of these studies was performed before in a sample of Portuguese population. The main aims of this study were i) to estimate the prevalence of obesity in 6-12 years old children from the central region of Portugal; ii) to investigate whether 14 previously described SNPs in obesity-related genes are associated with the risk of obesity in Portuguese children; iii) to identify MC4R gene mutations in children with morbid obesity (BMI ≥99th) that could justify this phenotype. Anthropometric parameters such as weight, height and waist circumference were measured in a random representative sample of 1433 children (747 girls and 686 boys) between 6-12 years old from several public schools in 2011. The International Obesity Task Force (IOTF) cut-offs were used to define obesity. Children classified with overweight (320) and obesity (154), and 256 randomly selected lean subjects with 18.5MI<25 kg/m2, were selected for genotyping. Polymorphisms were examined in DNA samples by TaqMan® assay. Sequencing of the MC4R gene was carried out in all individuals with BMI ≥99th. The prevalence of overweight/obesity found in the total sample was 33.0%; 10.7% were obese children. Overweight was significantly higher in boys than in girls (25.9% and 19.0% respectively, p=0.04), whereas no gender differences was found for obesity (10.6 % and 10.7% respectively, p=0.57). Comparison with previous studies showed a slightly increase in overweight/obesity in children of central Portugal in the last 10 years, reaching values of 40.0% prevalence in the 7-9 years old children. For three study polymorphisms located in the FTO gene, we found significant associations for SNPs rs9939609 and rs1421085 with weight, BMI, BMI Z-score and waist circumference (p<0.05 in all traits). For rs1861868, marginally significant associations were obtained with weight (p=0.08) and BMI (p=0.09). Logistic regression analysis, in the additive model, revealed both rs9939609 and rs1421085 SNPs significantly associated with obesity (OR=1.41; p=0.02 and OR=1.45, p=0.01, respectively). Haplotype analyses (rs1861868-rs1421085-rs9939609) identified two combinations (ACA and GCA) associated with a higher risk of obesity (OR=1.53; p=0.02 and OR=1.73; p=0.03, respectively). We tested the association between the -13910C>T polymorphism, located in the lactase (LCT) gene, and obesity-related traits, and found indication for an association between the -13910*T allele and abdominal obesity (OR=1.41; p=0.03). Under the dominant model, significant association was observed between the LCT-13910 CT/TT genotypes and abdominal obesity (OR=1.65; p=0.02). No association was detected with the risk of obesity (p=0.35) or anthropometrics traits (p>0.05). Finally, we tested for the association of obesity-related quantitative traits in Portuguese children with ten polymorphisms in the obesity related genes MSRA, TFAP2B, MC4R, NRXN3, PPARGC1A, TMEM18, SEC16B, HOXB5 and OLFM4. The MC4R rs12970134 polymorphism was nominally associated with BMI (p=0.03), BMI Z-score (p=0.04) and waist circumference (p=0.02), and borderline associated with weight (p=0.05). Near nominal associations were also found for the PPARGC1A rs8192678 polymorphism with weight (p=0.06), and for the MSRA rs545854 polymorphism with BMI (p=0.05) and BMI Z-score (p=0.05). Furthermore, logistic regression under the additive model showed that MC4R rs12970134 and TFAP2B rs987237 were nominally, respectively, associated (OR=1.47; p=0.02) and borderline associated (OR=1.47; p=0.05) with the obese phenotype. Additionally, 32 children who present a BMI ≥99th were screened for MC4R gene mutations. We found two previously described polymorphisms at heterozygous state in two children: the -174A>C (rs34114122) polymorphism in the 5´UTR region in a girl with BMI Z-score=2.51; and the common missense mutation 307G>A (Val103Ile) in the MC4R gene coding region identified in a boy with a BMI Z-score=2.60. No other pathogenic MC4R gene mutations were detected. In conclusion, this study shows a high prevalence of overweight/obesity among Portuguese children, following the trend of other European countries. We highlighted for the first time the possible association of FTO, MC4R, PPARGC1A, MSRA and TFAP2B SNPs with several obesity-related traits in a sample of Portuguese children. FTO SNPs showed the strong association with the risk of obesity in line with previous studies performed in European populations. Our study is a significant contribution to the knowledge of the genetic basis of obesity in the Portuguese population, but further studies are needed to a better understanding of the genetic factors linked to the obesity risk. Such information could be used in the future for the development of new obesity preventive strategies.

FCT - SFRH/BD/68774/2010