Association of the MC4R rs17782313 polymorphism with plasma ghrelin, leptin, IL6 and TNFα concentrations, food intake and eating behaviors in morbidly obese women.

PURPOSE: The rs17782313 variant of the MC4R gene plays an important role in the obesity phenotype. Studies that evaluate environmental factors and genetic variants associated with obesity may represent a great advance in understanding the development of this disease. This work seeks to assess the association of the polymorphism of MC4R rs17782313 on plasma parameters, including leptin, ghrelin, tumor necrosis factor (TNFα) and interleukin 6 (IL6), and on the eating behaviors of morbidly obese women. METHODS: 70 adult women with BMI between 40 and 60 kg/m2 were recruited. Laboratory and anthropometric data were recorded. Using a visual analog scale (VAS), the feelings of hunger and satiety were evaluated. The presence or absence of binge eating was evaluated through the Binge Eating Scale (BES) questionnaire. Habitual food intake was analyzed using 3-day dietary records. TaqMan® assays were conducted using real-time PCR to assess genotype polymorphism variants from peripheral blood DNA. RESULTS: This study found that female patients with the MC4R rs17782313 polymorphism had high levels of ghrelin and reduced levels of IL6 in the postprandial period. We observed a higher prevalence of severe binge eating in more than 50% of women with at least one risk allele. CONCLUSION: Our hypothesis is that the MC4R rs17782313 polymorphism may influence the release of ghrelin, even without being associated with feelings of hunger and satiety. More than half of women with this polymorphism exhibited severe binge eating. LEVEL OF EVIDENCE: Level III: case-control analytic study.

Study of LEP, MRAP2 and POMC genes as potential causes of severe obesity in Brazilian patients.

PURPOSE: Monogenic forms of obesity are caused by single-gene variants which affect the energy homeostasis by increasing food intake and decreasing energy expenditure. Most of these variants result from disruption of the leptin-melanocortin signaling, which can cause severe early-onset obesity and hyperphagia. These mutation have been identified in genes encoding essential proteins to this pathway, including leptin (LEP), melanocortin 2 receptor accessory proteins 2 (MRAP2) and proopiomelanocortin (POMC). We aimed to investigate the prevalence of LEP, MRAP2 and POMC rare variants in severely obese adults, who developed obesity during childhood. To the best of our knowledge, this is the first study screening rare variants of these genes in patients from Brazil. METHODS: A total of 122 Brazilian severely obese patients (BMI ≥ 35 kg/m2) were screened for the coding regions of LEP, MRAP2 and POMC by Sanger sequencing. All patients are candidates to the bariatric surgery. Clinical characteristics were described in patients with novel and/or potentially pathogenic variants. RESULTS: Sixteen different variants were identified in these genes, of which two were novel. Among them, one previous variant with potentially deleterious effect in MRAP2 (p.Arg125Cys) was found. In addition, two heterozygous mutations in POMC (p.Phe87Leu and p.Arg90Leu) were predicted to impair protein function. We also observed a POMC homozygous 9 bp insertion (p.Gly99_Ala100insSerSerGly) in three patients. No pathogenic variant was observed in LEP. CONCLUSION: Our study described for the first time the prevalence of rare potentially pathogenic MRAP2 and POMC variants in a cohort of Brazilian severely obese adults. LEVEL OF EVIDENCE: Level V, cross-sectional descriptive study.

Genetic variants in the fat mass and obesity-associated (FTO) gene confer risk for extreme obesity and modulate adiposity in a Brazilian population.

Obesity is a major public health problem worldwide. It has a complex etiology, influenced by environmental and genetic factors. FTO has been recognized as an important genetic factor for obesity development. This study evaluated the contribution of FTO polymorphisms (rs9939609 and rs17817449) for extreme obesity in terms of the period of obesity onset, anthropometric, and biochemical parameters. The haplotype and the combined effects of FTO risk alleles on obesity susceptibility were evaluated. We investigated 169 normal-weight subjects (body mass index, BMI: 22.8 [21.0; 24.0] kg/m2) and 123 extremely obese individuals (BMI: 47.6 [44.1; 53.1] kg/m2). Genotyping was performed by real time PCR. Our results showed a strong association between FTO variants and extreme obesity. Carriers of the AT haplotype had an increased risk for extreme obesity. Gene scores suggested that the risk of developing extreme obesity was increased 1.37-fold per risk allele added. Both polymorphisms also influenced BMI and body weight. Additionally, rs17817449 influenced triglyceride levels. No effect of FTO variants on the period of obesity onset was found. In conclusion, the FTO polymorphisms showed a strong association with development of extreme phenotype of obesity and adiposity modulation in a Brazilian population.

HLA-A*01 allele: a risk factor for dengue haemorrhagic fever in Brazil's population.

Severe forms of dengue, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome, are examples of a complex pathogenic mechanism in which the virus, environment and host immune response interact. The influence of the host's genetic predisposition to susceptibility or resistance to infectious diseases has been evidenced in several studies. The association of the human leukocyte antigen gene (HLA) class I alleles with DHF susceptibility or resistance has been reported in ethnically and geographically distinct populations. Due to these ethnic and viral strain differences, associations occur in each population, independently with a specific allele, which most likely explains the associations of several alleles with DHF. As the potential role of HLA alleles in the progression of DHF in Brazilian patients remains unknown, we then identified HLA-A alleles in 67 patients with dengue fever and 42 with DHF from Rio de Janeiro, Brazil, selected from 2002-2008 by the sequence-based typing technique. Statistical analysis revealed an association between the HLA-A*01 allele and DHF [odds ratio (OR) = 2.7, p = 0.01], while analysis of the HLA-A*31 allele (OR = 0.5, p = 0.11) suggested a potential protective role in DHF that should be further investigated. This study provides evidence that HLA class I alleles might be important risk factors for DHF in Brazilian patients.

Next-Generation Sequencing for Molecular Diagnosis of Cystic Fibrosis in a Brazilian Cohort.

Cystic fibrosis (CF), an autosomal recessive genetic disease, is recognized as one of the most prevalent diseases in Caucasian populations. Epidemiological data show that the incidence of CF varies between countries and ethnic groups in the same region. CF occurs due to pathogenic variants in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), located on chromosome 7q31.2. To date, more than 2,000 variants have been registered in the CFTR database. The study of these variants leads to the diagnosis and the possibility of a specific treatment for each patient through precision medicine. In this study, complete screening of CFTR was performed through next-generation sequencing (NGS) to gain insight into the variants circulating in the population of Rio de Janeiro and to provide patient access to treatment through genotype-specific therapies. Samples from 93 patients with an inconclusive molecular diagnosis were subjected to full-length screening of CFTR using an Illumina NGS HiSeq platform. Among these patients, 46 had two pathogenic variants, whereas 12 had only one CFTR variant. Twenty-four variants were not part of our routine screening. Of these 24 variants, V938Gfs∗37 had not been described in the CF databases previously. This research achieved a molecular diagnosis of the patients with CF and identification of possible molecular candidates for genotype-specific treatments.

A Rare Potential Pathogenic Variant in the BDNF Gene is Found in a Brazilian Patient with Severe Childhood-Onset Obesity.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a pro-survival factor in the brain that also regulates energy balance. BDNF loss-of-function point mutations are responsible for haploinsufficiency, causing severe early-onset obesity. Up to date, only a few studies have sequenced this gene to search for rare mutations related to obesity. In this study, we aimed to investigate the prevalence of BDNF variants in a cohort of adults with severe obesity from Brazil. MATERIAL AND METHODS: This study comprised 201 adults with severe obesity (BMI ≥ 35.0 kg/m2) with onset during childhood- or adolescence/youth. As controls, 73 subjects with normal weight (18.5 ≤ BMI ≤ 24.9 kg/m2) were selected. The exclusion criteria were pregnancy, lactation, the use of medication to lose or gain weight, and the presence of symptoms suggestive of syndromic obesity (only for the case group). The coding region of the BDNF gene was screened by Sanger sequencing. Demographic, anthropometric, and blood pressure parameters were obtained from the participants as well as serum hormone and cytokines concentrations and biochemical values. RESULTS: As a result, three missense variants [p.(Thr2Ile), p.(Val66Met), and p.(Arg209Gln)] and four synonymous variants (p.Leu107=, p.Thr149=, p.Ala150=, and p.Ser213=) were identified. The p.(Arg209Gln) was predicted as pathogenic by all in silico algorithms used and was not observed in the control group. The individuals carrying the p.(Val66Met) mutated allele had higher waist circumference, HDL-cholesterol and MCP1 levels, and reduced risk of developing metabolic syndrome. CONCLUSION: We observed that the common BDNF p.(Val66Met) variant has influenced waist circumference, HDL-cholesterol, and MCP1 levels. This polymorphism has also a protective effect on metabolic syndrome susceptibility. Additionally, we described for the first time a rare potentially pathogenic BDNF variant in a Brazilian patient with severe obesity and childhood-onset.

Genetic Variants in the Activation of the Brown-Like Adipocyte Pathway and the Risk for Severe Obesity.

BACKGROUND: Regular physical activity has an important role in energy expenditure and combats the development of obesity. During exercise, PPARGC1A is overexpressed, stimulating an increase of the expression of FNDC5. This protein is cleaved to release the hormone irisin, which activates a browning process in white adipose tissue through an increase in UCP1 expression. As a result, irisin leads to mitochondrial heat production and energy expenditure. OBJECTIVES: The aim of this study was to investigate whether genetic variants in genes related to browning are associated with severe obesity and obesity-related features. This case-control study comprised 210 individuals with severe obesity (median body mass index [BMI] 45.6 [range 40.5-52.2]) and 191 normal-weight subjects (BMI 22.8 [21.1-23.9]). METHODS: Genomic DNA was extracted from peripheral blood and the genotypes of the PPARGC1A(rs8192678, rs3736265, rs2970847, and rs3755863) and UCP1 (rs6536991 and rs12502572) genes were obtained using Taqman® assay. For the FNDC5 gene, screening of exons 3-5 as well as their intron-exon boundaries was performed using automatic sequencing. RESULTS: Our results demonstrated that PPARGC1Ars2970847 and UCP1rs12502572 are associated with severe obesity. Furthermore, these polymorphisms influence anthropometric traits, such as BMI, body weight, and body adiposity index. Our findings also showed a dose-effect relationship between PPARGC1A rs8192678 and fasting plasma glucose. Finally, 5 rare mutations were identified in FNDC5, and 1 of these is a novel missense mutation. CONCLUSION: This study shows that genetic variants in the activation of brown-like adipocyte pathway play an important role in the susceptibility to severe obesity.

Identification of a Rare and Potential Pathogenic MC4R Variant in a Brazilian Patient With Adulthood-Onset Severe Obesity.

BACKGROUND: The melanocortinergic pathway orchestrates the energy homeostasis and impairments in this system often lead to an increase in body weight. Rare variants in the melanocortin 4 receptor (MC4R) gene resulting in partial or complete loss of function have been described with autosomal co-dominant inheritance. These mutations are the most common cause of non-syndromic monogenic obesity. In this context, this study aimed to sequence the MC4R gene in a Brazilian cohort of adults with severe obesity. METHODS: This study included 163 unrelated probands with Body Mass Index (BMI) ≥ 35 kg/m2, stratified into three groups, according to the period of obesity onset. From the total sample, 25 patients were enrolled in the childhood-onset group (0-11 years), 19 patients in the adolescence/youth-onset group (12-21 years), and 119 patients in the adult-onset group (>21 years). Blood pressure, anthropometric and biochemical characteristics were obtained, and the MC4R coding region of each subject's DNA was assessed using automated Sanger sequencing. RESULTS: Significant anthropometric differences between the groups were observed. Higher body weight and BMI medians were found in patients with childhood-onset or adolescence/youth-onset when compared to the adulthood-onset obesity group. A total of five mutations were identified, including four missense variants: p.Ser36Thr, p.Val103Ile, p.Ala175Thr, and p.Ile251Leu. Additionally, we observed one synonymous variant (p.Ile198=). The p.Ala175Thr variant was identified in a female case with severe obesity and adulthood-onset. This variant was previously described as a partial loss-of-function mutation, in which the minor allele poses dominant-negative effect, probably resulting in reduced cAMP activity. CONCLUSION: This study showed a prevalence of common and rare variants in a cohort of Brazilian adults with severe obesity and candidates to bariatric surgery. We have identified a rare potentially pathogenic MC4R variant in a Brazilian patient with severe and adulthood-onset obesity.

The association of the fat mass and obesity-associated gene (FTO) rs9939609 polymorphism and the severe obesity in a Brazilian population.

Background: Obesity occurs due to the interaction between the genetic background and environmental factors, including an increased food intake and a sedentary lifestyle. Nowadays, it is clear that there is a specific circuit, called leptin-melanocortin pathway, which stimulates and suppresses food intake and energy expenditure. Therefore, the aim of this study was to evaluate the influence of genetic variants related to appetite regulation and energy expenditure on severe obesity susceptibility and metabolic phenotypes in a Brazilian cohort. Material and methods: A total of 490 participants were selected (298 severely obese subjects and 192 normal-weight individuals). Genomic DNA was extracted and polymorphisms in protein related to agouti (AGRP; rs5030980), ghrelin (GHRL; rs696217), neuropeptide Y (NPY; rs535870237), melanocortin 4 receptor (MC4R; rs17782313), brain-derived neurotrophic factor (BDNF; rs4074134) and fat mass and obesity-associated (FTO; rs9939609) genes were genotyped using TaqMan® probes. Demographic, anthropometric, biochemical and blood pressure parameters were obtained from the participants. Results: Our results showed that FTO rs9939609 was associated with severe obesity susceptibility. This polymorphism was also related to body weight, body mass index (BMI), waist to weight ratio (WWR) and inverted BMI. Individuals carrying the mutant allele (A) showed higher levels of BMI as well as lower values of WWR and inverted BMI. Conclusion: This study showed that FTO rs9939609 polymorphism plays a significant role in predisposing severe obesity in a Brazilian population.

Identification of a novel large deletion and other copy number variations in the CFTR gene in patients with Cystic Fibrosis from a multiethnic population.

BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). There are over 2000 different pathogenic and non-pathogenic variants described in association with a broad clinical heterogeneity. The most common types of mutations in this gene are single nucleotide substitutions or small deletions and insertions. However, large rearrangements, such as large duplications or deletions, are also a possible cause of CF; these variations are rarely tested in routine screenings, and much of them remain unidentified in some populations, especially those with high ethnic heterogeneity. METHODS: The present study utilized the Multiplex Ligation-dependent Probe Amplification (MLPA) technique for the detection of duplications and deletions in 165 CF patients from the Rio de Janeiro State (Brazil), which after extensive mutational screening, still exhibited one or two unidentified CF alleles. RESULTS: Five patients with alterations in MLPA signals were detected. After validation, we identified three copy number variations, one large duplication (CFTRdup2-3) and two large deletions (CFTRdel25-26 and CFTRdel25-27-CTTNBP2). Two detected deletions were not validated. They were false positives caused by a small deletion of 18 base pairs (232del18) and a point mutation (S168L) in the probe binding site. CONCLUSION: Our results highlight the importance of screening for large rearrangements in CF cases with no or only one CFTR mutation defined.

Identification of the MC4R start lost mutation in a morbidly obese Brazilian patient.

BACKGROUND: Melanocortin 4 receptor gene (MC4R) is an important regulator of food intake, body weight, and blood pressure. Mutations in MC4R are associated with the most common form of nonsyndromic monogenic obesity. MC4R variations have an autosomal co-/dominant model of inheritance. MC4R screening could reveal individuals previously unrecognized with Mendelian form of obesity for further clinical management and genetic counseling. However, there are limited data regarding MC4R variants in patients with obesity from Brazil. The aim of this study was to screen the coding region of the MC4R gene in a Brazilian cohort of severely obese adults and to investigate the phenotype-genotype correlation within MC4R variant carriers. METHODS: This study comprised 157 adult participants, stratified according to the period of obesity onset. The first group included 97 patients with childhood-onset obesity (0-11 years) and the second group comprised 60 subjects with adolescence/youth-onset obesity (12-21 years). The entire coding region of MC4R gene was screened by Sanger sequencing. RESULTS: As a result, five previously described variants (Met1?, Ser36Thr, Val103Ile, Ile98=, and Phe202Leu) were identified. Met1? is a start lost codon variant, which affects the translation of MC4R. It was found in a female patient with childhood-onset obesity. We also compared the anthropometric and metabolic parameters between patients with MC4R missense variants (Ser36Thr, Val103Ile, and Phe202Leu) and noncarriers. Patients carrying MC4R variants had higher median of waist-hip ratio when compared to noncarriers (P=0.048). These missense variants were also associated with hypertension (P=0.014). Additionally, Val103Ile carriers had lower diastolic blood pressure and lower systolic blood pressure compared to noncarriers (P=0.020 and P=0.065, respectively). Val103Ile was also associated with hypertension (P=0.003). CONCLUSION: This study showed the prevalence of MC4R variants in a cohort of Brazilian adults with severe obesity. We also identified significant phenotype differences between carriers and noncarriers of missense variants in our sample, suggesting an important role of MC4R on body fat distribution and blood pressure.

Influence of FTO rs9939609 polymorphism on appetite, ghrelin, leptin, IL6, TNFα levels, and food intake of women with morbid obesity.

BACKGROUND: The fat mass and obesity-related (FTO) gene has a strong relationship with obesity, extreme obesity and inflammatory state, and may also be associated with food intake regulation. OBJECTIVE: The aim of the present study was to evaluate the influence of the rs9939609 single-nucleotide polymorphism of the FTO gene on appetite, ghrelin, leptin, interleukin 6 (IL6), tumor necrosis factor α (TNFα) levels and food intake of morbidly obese women. MATERIALS AND METHODS: The study comprised 70 women, aged between 20 and 48 years, from Rio de Janeiro, Brazil. The participants were selected according to the body mass index between 40 and 60 kg/m2. Anthropometric and biochemical data were measured during fasting. Hormones and inflammatory data were measured before and after the participants ate an isocaloric meal. Dietary records were calculated and analyzed using a nutritional assessment program. Visual analog scales were used for behaviors of the sensations of appetite and food preferences. The FTO rs9939609 variant was genotyped using real-time polymerase chain reaction. RESULTS: Participants with the AA genotype had lower values of ghrelin and IL6 and higher values of leptin than those with TT and TA in the postprandial period. Comparing the plasma concentrations of ghrelin, insulin, IL6 and TNFα intragenotypes, it was observed that those with TT had decreased leptin and increased IL6 at the postprandial period. Subjects with TA showed increased postprandial IL6, and those with AA had decreased postprandial ghrelin. There was no difference in TNFα intra- and intergenotypes. The postprandial sensations of hunger were lower in AA than those with TT. There were differences between genotypes regarding ingested grams of protein by weight, cholesterol, B3, B5, B6 and B12 vitamins, and selenium potassium and sodium minerals. CONCLUSION: These findings suggest that genetics may exert an influence on physiologic factors and might alter eating behavior.

Adiponectin, Retinoic Acid Receptor Responder 2, and Peroxisome Proliferator-Activated Receptor-γ Coativator-1 Genes and the Risk for Obesity.

Obesity is the most common nutritional disorder. This disease is a multifactorial disease influenced by environmental and genetic factors. This study investigated the relationship between common variants of adiponectin (ADIPOQ), retinoic acid receptor responder 2 (RARRES2), and peroxisome proliferator-activated receptor-γ coativator-1 (PPARGC1) and obesity-related traits and susceptibility. A total of 167 individuals with obesity and 165 normal-weight subjects were recruited. Genotype frequencies of rs182052 in ADIPOQ differed significantly between the groups. Genotype AA was observed at a higher frequency in case than in control subjects. Association analysis showed that the A allele was a risk factor for obesity. This polymorphism was associated with body weight, body mass index (BMI), and waist circumference. After stratification by BMI, eutrophic individuals with AA or AG genotypes had higher body weights and waist circumferences than those with GG genotypes. In the case group, no associations were observed, except for stratified subjects with morbid obesity that exhibited a progressive increase of body weight, BMI, and waist circumference when rs182052 A was present. No associations were observed between SNPs in RARRES2 and PPARGC1 and obesity or any other studied variables. The rs182052 polymorphism in ADIPOQ is associated with a higher risk for obesity and obesity-related parameters.

Repeatability and Diagnostic Value of Nasal Potential Difference in a Genetically Admixed Population.

BACKGROUND: The genetic diversity of the Brazilian population results from three ethnic groups admixture: Europeans, Africans and Amerindians, thus increasing the difficulty of performing cystic fibrosis (CF) diagnosis. The nasal potential difference (NPD) evaluates the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) activity. Despite being a useful CF diagnostic test and a biomarker of CFTR-modulator drugs, it is also highly operator dependent. Therefore, it may be difficult to get accurate results and to interpret them. Wilschanski and Sermet scores were proposed to address these issues. This study aimed to evaluate repeatability and diagnostic value of NPD parameters and Wilschanski and Sermet scores in a CF center in Rio de Janeiro. METHODS: NPD was performed in 78 subjects. Maximal PD, amiloride response, total chloride response, and Wilschanski and Sermet scores were explored as means (confidence interval, CI). One-way ANOVA was used to compare mean differences and Scheffe test was used to pair-wise comparisons. Repeatability was evaluated by scatter and Bland-Altman plots. The Ethics Committee of the CF Center has approved the study protocol. Parents and adult participants signed an informed consent form. RESULTS: Forty-eight healthy-volunteers, 19 non-CF and 11 CF patients were enrolled in this study. Significant differences were found when comparing CF patients' NPD parameters to the other two groups (P = 0.000). Moreover, no significant differences were found when parameters from non-CF patients were compared with those from healthy volunteers (P > 0.05). The means of NPD parameters and diagnostic scores of each group were in concordance with disease/non-disease conditions. The repeatability data - Wilschanski and Sermet and NPD - allow NPD to be performed in this Brazilian CF Center. CONCLUSIONS: The present study gathered consistent data for Bland-Altman plots. The results of Wilschanski and Sermet diagnostic scores suggest that they were concordant with CF/non-CF conditions. More NPD tests should be performed in the Rio de Janeiro CF dynamic cohort to contribute to international NPD validation studies and to provide NPD as a biomarker in Brazil.

Severe phenotype in an apparent homozygosity caused by a large deletion in the CFTR gene: a case report.

BACKGROUND: Over 1900 mutations have been identified in the cystic fibrosis conductance transmembrane regulator gene, including single nucleotide substitutions, insertions, and deletions. Unidentified mutations may still lie in introns or in regulatory regions, which are not routinely investigated, or in large genomic deletions, which are not revealed by conventional molecular analysis. The apparent homozygosity for a rare, cystic fibrosis conductance transmembrane regulator mutation screened by standard molecular analysis should be further investigated to confirm if the mutation is in fact homozygous. We describe a patient presenting with an apparent homozygous S4X mutation. CASE PRESENTATION: A 13-year-old female patient of African descent with clinical symptoms of classic cystic fibrosis and a positive sweat test (97 mEq/L, diagnosed at age 3 years) presented with pancreatic insufficiency and severe pulmonary symptoms (initial lung colonization with Pseudomonas aeruginosa at age 4 years; forced vital capacity: 69%; forced expiratory volume: 51%; 2011). Furthermore, she developed severe acute lung disease and recurrent episodes of dehydration requiring hospitalization. The girl carried the CFTR mutation S4X in apparent homozygosity. However, further analysis revealed a large deletion in the second allele that included the region of the mutation. The deletion that we describe includes nucleotides 120-142, which correspond to a loss of 23 nucleotides that abolishes the normal translation initiation codon. CONCLUSION: This study reiterates the view that large, cystic fibrosis conductance transmembrane regulator deletions are an important cause of severe cystic fibrosis and emphasizes the importance of including large deletions/duplications in cystic fibrosis conductance transmembrane regulator diagnostic tests.