Genetic variants in DBC1, SIRT1, UCP2 and ADRB2 as potential biomarkers for severe obesity and metabolic complications.

Introduction: Obesity is a multifactorial disease associated with the development of many comorbidities. This disease is associated with several metabolic alterations; however, it has been shown that some individuals with obesity do not exhibit metabolic syndrome. Adipose tissue neutralizes the detrimental effects of circulating fatty acids, ectopic deposition, and inflammation, among others, through its esterification into neutral lipids that are stored in the adipocyte. However, when the adipocyte is overloaded, i.e., its expansion capacity is exceeded, this protection is lost, resulting in fatty acid toxicity with ectopic fat accumulation in peripheral tissues and inflammation. In this line, this study aimed to investigate whether polymorphisms in genes that control adipose tissue fat storage capacity are potential biomarkers for severe obesity susceptibility and also metabolic complications. Methods: This study enrolled 305 individuals with severe obesity (cases, BMI≥35 kg/m2) and 196 individuals with normal weight (controls, 18.5≤BMI≤24.9 kg/m2). Demographic, anthropometric, biochemical, and blood pressure variables were collected from the participants. Plasma levels of leptin, resistin, MCP1, and PAI1 were measured by Bio-Plex 200 Multiplexing Analyzer System. Genomic DNA was extracted and variants in DBC1 (rs17060940), SIRT1 (rs7895833 and rs1467568), UCP2 (rs660339), PPARG (rs1801282) and ADRB2 (rs1042713 and rs1042714) genes were genotyped by PCR allelic discrimination using TaqMan® assays. Results: Our findings indicated that SIRT1 rs7895833 polymorphism was a risk factor for severe obesity development in the overdominant model. SIRT1 rs1467568 and UCP2 rs660339 were associated with anthropometric traits. SIRT1 rs1467568 G allele was related to lower medians of body adipose index and hip circumference, while the UCP2 rs660339 AA genotype was associate with increased body mass index. Additionally, DBC1 rs17060940 influenced glycated hemoglobin. Regarding metabolic alterations, 27% of individuals with obesity presented balanced metabolic status in our cohort. Furthermore, SIRT1 rs1467568 AG genotype increased 2.5 times the risk of developing metabolic alterations. No statistically significant results were observed with Peroxisome Proliferator-Activated Receptor Gama and ADRB2 polymorphisms. Discussion/Conclusion: This study revealed that SIRT1 rs7895833 and rs1467568 are potential biomarkers for severe obesity susceptibility and the development of unbalanced metabolic status in obesity, respectively. UCP2 rs660339 and DBC1 rs17060940 also showed a significant role in obesity related-traits.

A Pilot Study of Dietetic, Phenotypic, and Genotypic Features Influencing Hypertensive Disorders of Pregnancy in Women with Pregestational Diabetes Mellitus.

Hypertensive disorders of pregnancy (HDP) are a leading cause of maternal and perinatal morbimortality. Dietetic, phenotypic, and genotypic factors influencing HDP were analyzed during a nutrigenetic trial in Rio de Janeiro, Brazil (2016-2020). Pregnant women with pregestational diabetes mellitus (n = 70) were randomly assigned to a traditional or DASH diet group. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured during prenatal visits and HDP were diagnosed using international criteria. Phenotypic data were obtained from medical records and personal interviews. Genotyping for FTO and ADRB2 polymorphisms used RT-PCR. Linear mixed-effect models and time-to-event analyses were performed. The variables with significant effect on the risk for progression to HDP were: black skin color (adjusted hazard ratio [aHR] 8.63, p = 0.01), preeclampsia in previous pregnancy (aHR 11.66, p < 0.01), SBP ≥ 114 mmHg in the third trimester (aHR 5.56, p 0.04), DBP ≥ 70 mmHg in the first trimester (aHR 70.15, p = 0.03), mean blood pressure > 100 mmHg (aHR 18.42, p = 0.03), and HbA1c ≥ 6.41% in the third trimester (aHR 4.76, p = 0.03). Dietetic and genotypic features had no significant effect on the outcome, although there was limited statistical power to test both.

SH2B1 variants as potential causes of non-syndromic monogenic obesity in a Brazilian cohort.

PURPOSE: SH2B1 gene encodes an important adaptor protein to receptor tyrosine kinases or cytokine receptors associated with Janus kinases. This gene has been associated with the structural and functional modulation of neurons and other cells, and impacts on energy and glucose homeostasis. Several studies suggested that alterations in this gene are strong candidates for the development of obesity. However, only a few studies have screened SH2B1 point variants in individuals with obesity. Therefore, the aim of this study was to investigate the prevalence of SH2B1 variants in a Brazilian cohort of patients with severe obesity and candidates to bariatric surgery. METHODS: The cohort comprised 122 individuals with severe obesity, who developed this phenotype during childhood. As controls, 100 normal-weight individuals were included. The coding region of SH2B1 gene was screened by Sanger sequencing. RESULTS: A total of eight variants were identified in SH2B1, of which p.(Val345Met) and p.(Arg630Gln) variants were rare and predicted as potentially pathogenic by the in the silico algorithms used in this study. The p.(Val345Met) was not found in either the control group or in publicly available databases. This variant was identified in a female patient with severe obesity, metabolic syndrome and hyperglycemia. The p.(Arg630Gln) was also absent in our control group, but it was reported in gnomAD with an extremely low frequency. This variant was observed in a female patient with morbid obesity, metabolic syndrome, hypertension and severe binge-eating disorder. CONCLUSION: Our study reported for the first time two rare and potentially pathogenic variants in Brazilian patients with severe obesity. Further functional studies will be necessary to confirm and elucidate the impact of these variants on SH2B1 protein function and stability, and their impact on energetic metabolism. LEVEL OF EVIDENCE: Level V, cross-sectional descriptive study.

Fibromodulin Gene Variants (FMOD) as Potential Biomarkers for Prostate Cancer and Benign Prostatic Hyperplasia.

By the year 2050, the world's elderly population may increase exponentially, raising the rate of disease characteristic of this group, such as prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Prostate disorders have a multifactorial etiology, especially age and genetic factors. Currently, PCa is the second most frequent neoplasm in the male population worldwide. The fibromodulin gene encodes a small leucine-rich proteoglycan (SLRP) which acts in the collagen fibrillogenesis pathway, cell adhesion, and modulation of TGF-ß signaling pathways, which has been recently associated with PCa. The present study sequenced the coding region of the FMOD in a sample of 44 PCa, 90 BPH, and 82 controls from a Brazilian population, and the results identified 6 variants: 2 missenses (p.(Tyr42Ser) and p.(Pro24Ala)); 3 synonymous (p.(His253=), p.(Asn353=), and p.(Glu79=)); and 1 intronic (c.980-114A>G). Of these, p.(Tyr42Ser), p.(Pro24Ala), and p.(Asn353=) are rare variants, and p.(Tyr42Ser) was predicted as potential pathogenic by the algorithms used here, in addition to not being observed in controls, suggesting that may be a potential biomarker for development of PCa and BPH. In conclusion, we identified for the first time, in Brazilian individuals with PCa and BPH, a potentially pathogenic variant in the analysis of FMOD gene. Further studies are needed to investigate the deleterious effect of this variant on the structure and/or function of the FMOD protein.

Identification of Variants Responsible for Monogenic Forms of Diabetes in Brazil.

Monogenic forms of diabetes mellitus may affect a significant number of patients of this disease, and it is an important molecular cause to be investigated. However, studies of the genetic causes of monogenic diabetes, especially in populations with mixed ethnic backgrounds, such as the one in Brazil, are scarce. The aim of this study was to screen several genes associated with monogenic diabetes in fifty-seven Brazilian patients with recurrence of the disease in their families and thirty-four relatives. Inclusion criteria were: Age of onset ≤ 40 years old, BMI < 30 kg/m², at least two affected generations and negative anti-GAD and anti-IA2 antibodies. MODY genes HNF4A, GCK, HNF1A, HNF1B, NEUROD1, KLF11, PAX4, INS, KCNJ11, and MT-TL1 were sequenced by Sanger sequencing. We identified a total of 20 patients with variants, 13 GCK-MODY, four HNF1A-MODY, and one variant in each of the following genes, HNF4A, HNF1B and MT-TL1. Segregation analysis was performed in 13 families. Four variants were novel, two in GCK (p.(Met115Val) [c.343A>G] and p.(Asp365GlufsTer95) [c.1094_1095insGCGA]) and two in HNF1A (p.(Tyr163Ter) [c.489C>G] and p.(Val380CysfsTer39) [c.1136_1137insC]). Here we highlight the importance of screening for monogenic diabetes in admixed populations.

FTO and ADRB2 Genetic Polymorphisms Are Risk Factors for Earlier Excessive Gestational Weight Gain in Pregnant Women with Pregestational Diabetes Mellitus: Results of a Randomized Nutrigenetic Trial.

Excessive gestational weight gain (GWG) is associated with increased risk of maternal and neonatal complications. We investigated obesity-related polymorphisms in the FTO gene (rs9939609, rs17817449) and ADRB2 (rs1042713, rs1042714) as candidate risk factors concerning excessive GWG in pregnant women with pregestational diabetes. This nutrigenetic trial, conducted in Brazil, randomly assigned 70 pregnant women to one of the groups: traditional diet (n = 41) or DASH diet (n = 29). Excessive GWG was the total weight gain above the upper limit of the recommendation, according to the Institute of Medicine guidelines. Genotyping was performed using real-time PCR. Time-to-event analysis was performed to investigate risk factors for progression to excessive GWG. Regardless the type of diet, AT carriers of rs9939609 (FTO) and AA carriers of rs1042713 (ADRB2) had higher risk of earlier exceeding GWG compared to TT (aHR 2.44; CI 95% 1.03-5.78; p = 0.04) and GG (aHR 3.91; CI 95% 1.12-13.70; p = 0.03) genotypes, respectively, as the AG carriers for FTO haplotype rs9939609:rs17817449 compared to TT carriers (aHR 1.79; CI 95% 1.04-3.06; p = 0.02).

When Leptin Is Not There: A Review of What Nonsyndromic Monogenic Obesity Cases Tell Us and the Benefits of Exogenous Leptin.

Obesity is a pandemic condition of complex etiology, resulting from the increasing exposition to obesogenic environmental factors combined with genetic susceptibility. In the past two decades, advances in genetic research identified variants of the leptin-melanocortin pathway coding for genes, which are related to the potentiation of satiety and hunger, immune system, and fertility. Here, we review cases of congenital leptin deficiency and the possible beneficial effects of leptin replacement therapy. In summary, the cases presented here show clinical phenotypes of disrupted bodily energy homeostasis, biochemical and hormonal disorders, and abnormal immune response. Some phenotypes can be partially reversed by exogenous administration of leptin. With this review, we aim to contribute to the understanding of leptin gene mutations as targets for obesity diagnostics and treatment strategies.

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.

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.

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.

Identification of the First PAX4-MODY Family Reported in Brazil.

PURPOSE: The aim of this study was to sequence the coding region of the PAX4 gene in a Brazilian cohort with clinical manifestations of monogenic diabetes. PATIENTS AND METHODS: This study included 31 patients with autosomal dominant history of diabetes, age at diagnosis ≤40 years, BMI <30 kg/m2, and no mutations in GCK or HNF1A, HNF4A, and HNF1B. Screening of the PAX4 coding region was performed by Sanger sequencing. In silico algorithms were used to assess the potential impact of amino acid substitutions on protein structure and function. Additionally, PAX4-MODY family members and 158 control subjects without diabetes were analyzed for the identified mutation. RESULTS: The molecular analysis of PAX4 has detected one missense mutation, p.Arg164Gln (c.491G>A), segregating with diabetes in a large Brazilian family. The mutation was absent among the control group. The index case is a woman diagnosed at 32 years of age with polyneuropathy and treated with insulin. She did not present diabetic renal disease or retinopathy. Family members with the PAX4 p.Arg164Gln mutation have a heterogeneous clinical manifestation and treatment response, with age at diagnosis ranging from 24 years to 50 years. CONCLUSION: To the best of our knowledge, this is the first study to report a PAX4-MODY family in Brazil. The age of PAX4-MODY diagnosis in the Brazilian family seems to be higher than the classical criteria for MODY. Our results reinforce the importance of screening large monogenic diabetes families for the understanding of the clinical manifestations of rare forms of diabetes for the specific and personalized treatment.

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.

MODY probability calculator for GCK and HNF1A screening in a multiethnic background population

ABSTRACT Objective We aimed to identify the frequency of monogenic diabetes, which is poorly studied in multiethnic populations, due to GCK or HNF1A mutations in patients with suggestive clinical characteristics from the Brazilian population, as well as investigate if the MODY probability calculator (MPC) could help patients with their selection. Subjects and methods Inclusion criteria were patients with DM diagnosed before 35 years; body mass index < 30 kg/m2; negative autoantibodies; and family history of DM in two or more generations. We sequenced HNF1A in 27 patients and GCK in seven subjects with asymptomatic mild fasting hyperglycemia. In addition, we calculated MODY probability with MPC. Results We identified 11 mutations in 34 patients (32.3%). We found three novel mutations. In the GCK group, six cases had mutations (85.7%), and their MODY probability on MPC was higher than 50%. In the HNF1A group, five of 27 individuals had mutations (18.5%). The MPC was higher than 75% in 11 subjects (including all five cases with HNF1A mutations). Conclusion Approximately one third of the studied patients have GCK or HNF1A mutations. Inclusion criteria included efficiency in detecting patients with GCK mutations but not for HNF1A mutations (< 20%). MPC was helpful in narrowing the number of candidates for HNF1A screening.

MODY probability calculator for GCK and HNF1A screening in a multiethnic background population.

Objective We aimed to identify the frequency of monogenic diabetes, which is poorly studied in multiethnic populations, due to GCK or HNF1A mutations in patients with suggestive clinical characteristics from the Brazilian population, as well as investigate if the MODY probability calculator (MPC) could help patients with their selection. Subjects and methods Inclusion criteria were patients with DM diagnosed before 35 years; body mass index < 30 kg/m2; negative autoantibodies; and family history of DM in two or more generations. We sequenced HNF1A in 27 patients and GCK in seven subjects with asymptomatic mild fasting hyperglycemia. In addition, we calculated MODY probability with MPC. Results We identified 11 mutations in 34 patients (32.3%). We found three novel mutations. In the GCK group, six cases had mutations (85.7%), and their MODY probability on MPC was higher than 50%. In the HNF1A group, five of 27 individuals had mutations (18.5%). The MPC was higher than 75% in 11 subjects (including all five cases with HNF1A mutations). Conclusion Approximately one third of the studied patients have GCK or HNF1A mutations. Inclusion criteria included efficiency in detecting patients with GCK mutations but not for HNF1A mutations (< 20%). MPC was helpful in narrowing the number of candidates for HNF1A screening.

The first case of NEUROD1-MODY reported in Latin America.

BACKGROUND: MODY-NEUROD1 is a rare form of monogenic diabetes caused by mutations in Neuronal differentiation 1 (NEUROD1). Until now, only a few cases of MODY-NEUROD1 have been reported worldwide and the real contribution of mutations in NEUROD1 in monogenic diabetes and its clinical impact remain unclear. METHODS: Genomic DNA was isolated from peripheral blood lymphocytes of 25 unrelated Brazilians patients with clinical characteristics suggestive of monogenic diabetes and the screening of the entire coding region of NEUROD1 was performed by Sanger sequencing. RESULTS: We identified one novel frameshift deletion (p.Phe256Leufs*2) in NEUROD1 segregating in an autosomal dominant inheritance fashion. Almost 20 years after the first report of NEUROD1-MODY, only a few families in Europe and Asia had shown mutations in NEUROD1 as the cause of monogenic diabetes. CONCLUSION: To our knowledge, we described the first case of NEUROD1-MODY in a Latin American family.

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.

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 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.

Abordagem epidemiológica e molecular da obesidade em uma amostra do Rio de Janeiro / Epidemiological and molecular approach to obesity in a sample from Rio de Janeiro

A obesidade é definida como um acúmulo de gordura no corpo (Índice de massa corpóreo, IMC ≥ 30 kg/m2), suficiente para causar efeitos adversos ao organismo. Essa doença tem emergido como um sério problema de saúde pública, afetando países desenvolvidos e em desenvolvimento. Dados recentes mostram que mais de 650 milhões de indivíduos são obesos no mundo. No Brasil, existiam pelo menos 11,9 milhões de homens e 18 milhões de mulheres com obesidade em 2014. Estudos epidemiológicos sugerem que indivíduos com alto IMC, como os obesos graves (IMC ≥ 35 kg/m2) e os mórbidos (IMC ≥ 40 kg/m2), possuem um risco substancialmente maior para o desenvolvimento de doenças associadas ao excesso do peso e morte prematura. A etiologia da obesidade é complexa, causada pela interação de fatores ambientais com variantes genéticas de pequeno efeito. No entanto, formas raras monogênicas foram identificadas em humanos, causadas por mutações de grande efeito em um único gene. Nos últimos anos, diversos genes têm sido associados à obesidade humana, podendo estar relacionados às formas poligênicas, monogênicas ou ambas. Dentre estes, podemos destacar os que atuam no sistema leptina-melanocortina e no mecanismo de escurecimento do tecido adiposo branco (browning). Estas vias estão envolvidas na homeostasia energética, controlando a ingestão alimentar e/ou o gasto energético do corpo. Dessa forma, o presente estudo tem como objetivo analisar variantes em genes associados a essas vias em uma coorte da população brasileira. Para esta proposta, selecionamos 496 indivíduos, divididos em um grupo de obesos graves (n=304) e outro de eutróficos (n=192, 18.5 ≤ IMC ≤ 24.9 kg/m2). Todos esses pacientes com excesso de peso são candidatos à cirurgia bariátrica. Para o estudo da obesidade monogênica selecionamos uma subamostra do nosso grupo de casos

Inicialmente, obtivemos as características demográficas, antropométricas, pressóricas e bioquímicas dos participantes. Em seguida, coletamos amostras de sangue e extraímos o DNA genômico. As análises moleculares foram realizadas através da reação em cadeia da polimerase (PCR) em tempo real (ensaios TaqMan®), PCR convencional e sequenciamento de Sanger. Nossos resultados mostraram que os obesos graves apresentam altos indicadores antropométricos, bioquímicos e pressóricos, com exceção da altura, IMC invertido e razão cintura-altura. Ademais, a prevalência de hipertensão arterial sistêmica (HAS) e diabetes mellitus tipo 2 (DM2) foram maiores nesse pacientes, assim como observamos um aumento da coloração da pele parda/negra e um estilo de vida sedentário. Com relação às análises moleculares, descrevemos pela primeira vez mutações patogênicas no gene MC4R (rs1356972268; p.Met1?) e MRAP2 (rs148904867; p.Arg125Cis). Também identificamos possíveis mutações com efeitos deletérios no FNDC5 (rs1249107410; p.Ser210Asn) e POMC (rs199636726; p.Fen87Leu e não descrita; p.Arg90Leu). Nenhuma variação genética patogênica foi encontrada em LEP. Além disso, observamos que polimorfismos no FTO (rs9939609 e rs17817449), PPARGC1A (rs2970847) e UCP1 (rs12502572) modulam a susceptibilidade à forma comum da obesidade e também influenciam a variação da adiposidade na amostra total. O FTO (rs17817449) e PPARGC1A (rs8192678) foram associados ao aumento das concentrações plasmáticas do triglicerídeo e glicose, respectivamente. O MC4R (rs2229616) mostrou-se um fator de proteção para HAS em obesos. Para finalizar, observamos que as variantes AGRP (rs5030980), BDNF (rs4074134), FNDC5 (rs113173936, rs72882318 e rs1284368), GHRL (rs696217), MC4R (rs17782313), NPY (rs535870237), PPARGC1A (rs3736265, rs3755863), UPC1 (rs6536991) não foram associadas à forma comum da obesidade, bem como não tinham um efeito nas variáveis estudadas

Portanto, nossos dados sugerem que determinadas variantes em genes importantes para as vias leptina-melanocortina e browning contribuem para o desenvolvimento da obesidade grave em uma amostra da população brasileira. (AU)