Sickle cell trait in São Tomé e Príncipe: a population-based prevalence study in women of reproductive age.

BACKGROUND: Sickle Cell Disorder is Africa's most prevalent genetic disease. Yet, it remains a neglected condition, with high mortality under-five, and a lack of population-based studies in the region. This is the first of its kind in São Tomé e Príncipe, aiming to estimate the prevalence of sickle cell trait and other haemoglobin variants in women of reproductive age and its associated factors. METHODS: We conducted a cluster survey in 35 neighbourhoods. Haemoglobin was assessed through point-of-care capillary electrophoresis or high-performance liquid chromatography, and sociodemographic data through questionnaires. The weighted prevalence of sickle cell trait (HbAS) and HbC carriers was estimated with a 95% confidence interval (95% CI). We calculated weighted prevalence ratios (95% CI) through robust Poisson regression for its association with age and individual and collective genetic heritage. FINDINGS: The prevalence of sickle cell trait in women of reproductive age in São Tomé e Príncipe (n = 376) was 13.45% (95% CI: 9.05-19.00). The prevalence of HbC carriers was 8.00% (95% CI: 4.71-12.00). Older age and speaking Forro or Angolar were positively associated with having sickle cell trait. INTERPRETATION: The prevalence of sickle cell trait in São Tomé e Príncipe ranks high in the West African region. The country should follow international guidelines, implementing newborn screening and comprehensive healthcare management.

Protective Association of APOC1/rs4420638 with Risk of Obesity: A case-control Study in Portuguese Children.

The association of the rs4420638 polymorphism, near the APOC1 gene, was examined with the risk of obesity among Portuguese children. A sample of 446 Portuguese individuals (231 boys and 215 girls) of European descent, aged 3.2 to 13.7 years old (mean age: 7.98 years), were selected to conduct a case-control study. Body mass index (BMI), BMI Z-scores, and waist circumference were calculated. Genotyping was performed by real time PCR using a pre-designed TaqMan probe. Logistic regression and the nonparametric Mann-Whitney test were used to test the associations. The association results revealed a significant protective effect from the minor G-allele of SNP rs4420638 against obesity, with an odds ratio (OR) of 0.619 (95% CI 0.421-0.913; p = 0.0155) in the additive model, and OR of 0.587 (95% CI 0.383-0.9; p = 0.0145) in the dominant model. Moreover, comparing genotype groups (AA vs. AG + GG), significantly lower values (p < 0.05) for the anthropometric traits weight, height, BMI, BMI Z-score and waist circumference, were observed in the carriers of allele G. The present study provides further evidence for the APOE/APOC1 candidate-region association with the risk of obesity. This was the first study to describe the protective association of the rs4420638 minor G-allele against obesity in childhood exclusively.

Differential sex-association between PCSK1 polymorphisms and obesity risk in Portuguese children.

OBJECTIVES: The proprotein convertase subtilisin/Kexin type 1 gene (PCSK1) is implicated in hypothalamic appetite control. Several studies have addressed the relationship between PCSK1 polymorphisms and obesity, although conflicting results were observed. We tested the potential association of four PCSK1 variants with the risk of overweight/obesity and related variables in Portuguese children. METHODS: This is a case-control study, where four PCSK1 variants, rs6230 (c.-101T>C), rs6232 (p.N221D), rs6235 (p.S690T), and rs3811942 (c.*265T>C), were analyzed in Portuguese children (aged 5-13 years-old). Anthropometric measures were objectively collected and used to provide weight-for-age, height-for-age, and body mass index (BMI) for age. The indices generated were compared to standard reference values of WHO to obtain the corresponding Z-scores. RESULTS: Logistic regression, in the dominant model, revealed no significant associations between the four individual PCSK1 variants and the risk of overweight/obesity in the total population. However, stratifying the sample by sex, a marginally significant association was found between the rs6235 minor C-allele and increased overweight/obesity in boys (n = 345) (OR 1.55 [1.01-2.38] p = .044), but not in girls (n = 340) (OR 0.73 [0.46-1.14] p = .169). Consistently, boys with genotype GG presented lower BMI Z-score (0.62) when compared to those with the genotypes GC + CC (1.04). Testing for different effects in males versus females, a significant interaction was found between the rs6235 polymorphism and sex for BMI Z-score (p = .025). CONCLUSIONS: Results of this study suggest for a sex-differentiated association between PCSK1 rs6235 and overweight/ obesity in Portuguese children.

Hb F Levels in ß-Thalassemia Carriers and Normal Individuals: Known and Unknown Quantitative Trait Loci in the ß-Globin Gene Cluster.

In the already identified quantitative trait loci (QTL), modulating Hb F levels are cis-acting haplotypes of the ß-globin gene cluster itself, although the single nucleotide polymorphisms (SNPs) accounting more for the association, remain uncertain. In this study, the role in Hb F production of previously reported candidate SNPs within the ß-globin gene cluster was reexamined, along with a yet poorly studied variation in the BGLT3 gene. In a sample of ß-thalassemia (ß-thal) carriers, we succeeded in replicating the significant association between increased Hb F levels and rs7482144 (C>T) (HBG2 XmnI), which is the most well-established variation in the cluster influencing the trait. This SNP was found to be in strong linkage disequilibrium (LD) with a variation in the HBBP1 gene [rs10128556 (G>A)], which consistently revealed a similar association signal. Remarkably, much stronger than the latter associations were those involving both rs968857 (T allele) (3' HBBP1) and rs7924684 (G allele) (BGLT3), two SNPs that were also in strong LD. As the pattern of LD detected in the ß-globin gene cluster does not correlate with a tight linkage between markers, complex interactions between SNPs at the cluster seem to modulate Hb F. Seeing that no such associations were detected in normal subjects, the question can be raised on whether, under erythropoiesis stress, epigenetic mechanisms contribute to change the regulation of the entire ß-globin gene cluster. In conclusion, we provide statistical evidence for a new player within the ß-globin gene cluster, BGLT3, that in cooperation with other regions influences Hb F levels in ß-thal carriers.

The population genomics of archaeological transition in west Iberia: Investigation of ancient substructure using imputation and haplotype-based methods.

We analyse new genomic data (0.05-2.95x) from 14 ancient individuals from Portugal distributed from the Middle Neolithic (4200-3500 BC) to the Middle Bronze Age (1740-1430 BC) and impute genomewide diploid genotypes in these together with published ancient Eurasians. While discontinuity is evident in the transition to agriculture across the region, sensitive haplotype-based analyses suggest a significant degree of local hunter-gatherer contribution to later Iberian Neolithic populations. A more subtle genetic influx is also apparent in the Bronze Age, detectable from analyses including haplotype sharing with both ancient and modern genomes, D-statistics and Y-chromosome lineages. However, the limited nature of this introgression contrasts with the major Steppe migration turnovers within third Millennium northern Europe and echoes the survival of non-Indo-European language in Iberia. Changes in genomic estimates of individual height across Europe are also associated with these major cultural transitions, and ancestral components continue to correlate with modern differences in stature.

Multi-Locus Models to Address Hb F Variability in Portuguese ß-Thalassemia Carriers.

Hb F production is under the influence of major quantitative trait loci (QTL). The present study aims: i) to replicate the association with Hb F for representative genetic variants in the three major Hb F QTLs in a Portuguese sample of ß-thalassemia (ß-thal) carriers; and ii) to test different genetic multi-locus models to account for the genetic component of Hb F variation. A population sample of 79 Portuguese ß-thal carriers (39 males, 40 females), aged between 2 to 70 years old, were genotyped for polymorphisms in the locus control region (LCR)-5' hypersensitive site 4 (5'HS4) rs16912979, XmnI-HBG2 rs7482144, BCL11A rs1427407 and HMIP rs66650371, using standard biomolecular procedures. Univariate linear regression models were used to test for genetic associations with Hb F. The minor alleles of the individual variants BCL11A rs1427407 (T) (0.165), HMIP rs66650371 (3 bp del) (0.247) and XmnI-HBG2 rs7482144 (T) (0.196), were found to be significantly associated with increased levels of Hb F (p = 0.029, p = 0.002 and p = 0.0004, respectively), explaining about 6.0, 12.0 and 15.0% of Hb F variation, respectively. In a multiple linear regression approach, the three loci accounted for about 30.0% of Hb F variance. Two genetic risk scores (GRS), rationalizing the number of minor alleles into a single genetic variable, explained about 30.0 and 32.0% of the Hb F variation. In conclusion, we replicated in ß-thal carriers previously reported associations with Hb F. Multi-locus models combining three representative variants of Hb F influencing QTLs can explain a larger amount of Hb F variability.

Physical activity and the association between the FTO rs9939609 polymorphism and obesity in Portuguese children aged 3 to 11 years.

OBJECTIVES: To investigate whether objectively measured physical activity (PA) modulates the association between the FTO rs9939609 polymorphism and obesity variables in a sample of Portuguese children. METHODS: A total of 440 children (213 girls and 227 boys) aged 3 to 11 years were observed. Genotyping was performed using TaqMan assay. Body mass index (BMI), BMI Z scores, waist circumference (WC), and waist-to-height ratio (WHtR) were calculated. PA was estimated in 399 children by accelerometry. RESULTS: Linear regression, in the additive model, showed that the rs9939609 minor A-allele significantly associated with BMI (P = .029), BMI Z score (P = .017), WC (P = .016), and WHtR (P = .019). Logistic regression, in the additive model, showed a marginally significant association between the A-allele and overweight/obesity (odds ratio [OR]: 1.372; P = .049). When stratified by sex, rs9939609 showed marginal or significant associations with BMI (P = .08), BMI Z score (P = .07), WC (P = .005), WHtR (P = .02), and overweight/obesity (OR: 1.529; P = .064) in girls but not in boys (P > .05). Significant interactions were not found between the FTO polymorphism and PA (inactive vs active groups of children) for BMI (P = .461), BMI Z score (P = .387), WC (P = .757), or WHtR (P = .621). CONCLUSIONS: Findings of the present study highlight the association between FTO rs9939609 and obesity or body fat indices in girls but not in boys. PA was not found to mediate the impact of FTO genetic variation on risk of obesity.

The Eastern side of the Westernmost Europeans: Insights from subclades within Y-chromosome haplogroup J-M304.

OBJECTIVES: We examined internal lineages and haplotype diversity in Portuguese samples belonging to J-M304 to improve the spatial and temporal understanding of the introduction of this haplogroup in Iberia, using the available knowledge about the phylogeography of its main branches, J1-M267 and J2-M172. METHODS: A total of 110 males of Portuguese descent were analyzed for 17 Y-chromosome bi-allelic markers and seven Y-chromosome short tandem repeats (Y-STR) loci. RESULTS: Among J1-M267 individuals (n = 36), five different sub-haplogroups were identified, with the most common being J1a2b2-L147.1 (∼72%), which encompassed the majority of representatives of the J1a2b-P58 subclade. One sample belonged to the rare J1a1-M365.1 lineage and presented a core Y-STR haplotype consistent with the Iberian settlement during the fifth century by the Alans, a people of Iranian heritage. The analysis of J2-M172 Portuguese males (n = 74) enabled the detection of the two main subclades at very dissimilar frequencies, J2a-M410 (∼80%) and J2b-M12 (∼20%), among which the most common branches were J2a1(xJ2a1b,h)-L26 (22.9%), J2a1b(xJ2a1b1)-M67 (20.3%), J2a1h-L24 (27%), and J2b2-M241 (20.3%). CONCLUSIONS: While previous inferences based on modern haplogroup J Y-chromosomes implicated a main Neolithic dissemination, here we propose a later arrival of J lineages into Iberia using a combination of novel Portuguese Y-chromosomal data and recent evidence from ancient DNA. Our analysis suggests that a substantial tranche of J1-M267 lineages was likely carried into the Iberian Peninsula as a consequence of the trans-Mediterranean contacts during the first millennium BC, while most of the J2-M172 lineages may be associated with post-Neolithic population movements within Europe.

High AMY1 copy number protects against obesity in Portuguese young adults.

BACKGROUND: The highly polymorphic copy number variation (CNV) in the salivary amylase gene (AMY1) has been associated with obesity in different populations. However, some authors have failed to reproduce these findings. AIM: To investigate the association between AMY1 CNV and obesity in young adults of Portuguese origin. SUBJECTS AND METHODS: This study evaluated AMY1 gene copy number (CN) in 262 individuals: 155 females and 107 males, aged 18-34 years-old (mean age = 21.08). The number of AMY1 copies was estimated in a QX100 droplet digital PCR (ddPCR) system (Bio-Rad Laboratories, Hercules, CA). RESULTS: Defining a case group with obese and overweight individuals, logistic regression did not show a significant association between AMY1 CNV and risk of overweight/obesity in the whole population (p = 0.489). However, after testing case-control data in the sub-set of samples above the third quartile (CN ≥10), a significant association was found between lower AMY1 copy number and risk of obesity (OR = 0.532; p = 0.034), even when adjusted for age and sex (OR = 0.527; p = 0.039). In concordance, all participants with >10 AMY1 copies were normal weight controls (n = 20) or overweight (n = 6). CONCLUSION: The results suggest that high AMY1 gene copy number protects against obesity in Portuguese young adults.

Polymorphisms in the SNRPN gene are associated with obesity susceptibility in a Spanish population.

BACKGROUND: SNRPN, which codes for the RNA-binding SmN protein, is a candidate gene for Prader-Willi syndrome. One characteristic of this neuroendocrine disorder is hyperphagia resulting in extreme obesity later in life. In the present study, we aimed to assess whether variability within this gene could be implicated in obesity susceptibility. METHODS: A case-control study was performed including 265 unrelated patients with nonsyndromic and early-onset severe obesity, belonging to high-risk obesity families from Spanish ancestry; 184 healthy control individuals were included representative of the same genetic background and sex-matched. Forty-nine single nucleotide polymorphisms (SNPs) spanning the entire SNRPN gene were selected and genotyped using the Sequenom MassARRAY platform (Sequenom Inc., San Diego, CA, USA). RESULTS: The four SNPs, rs12905653, rs752874, rs1391516 and rs2047433, were found to be nominally associated with obesity (p < 0.03). The diversity haplotype distribution among cases and controls identified the combination rs12905653-T/rs8028366-A/rs4028395-T as being strongly and inversely associated with obesity (odds ratio = 0.49; p = 0.0006). A genetic risk score was built based on rs12905653, rs1391516 and rs2047433 SNPs and each unit increase in genetic risk score increased the obesity risk by 49% (odds ratio = 1.49, 95% confidence interval = 1.24-1.80). CONCLUSIONS: To our knowledge, this is the first study reporting an association between variability in the SNRPN gene and the risk of being obese. Interestingly, it was the major allele of each SNP that was found to be associated with the risk of weight gain. Further studies analyzing this locus and the possible additive deleterious capability of SNP combinations could be useful for demonstrating the development of obesity.

The contribution of genetics and environment to obesity.

Background: Obesity is a global health problem mainly attributed to lifestyle changes such as diet, low physical activity or socioeconomics factors. However, several evidences consistently showed that genetics contributes significantly to the weight-gain susceptibility. Sources of data: A systematic literature search of most relevant original, review and meta-analysis, restricted to English was conducted in PubMed, Web of Science and Google scholar up to May 2017 concerning the contribution of genetics and environmental factors to obesity. Areas of agreement: Several evidences suggest that obesogenic environments contribute to the development of an obese phenotype. However, not every individual from the same population, despite sharing the same obesogenic environment, develop obesity. Areas of controversy: After more than 10 years of investigation on the genetics of obesity, the variants found associated with obesity represent only 3% of the estimated BMI-heritability, which is around 47-80%. Moreover, genetic factors per se were unable to explain the rapid spread of obesity prevalence. Growing points: The integration of multi-omics data enables scientists having a better picture and to elucidate unknown pathways contributing to obesity. Areas timely for developing research: New studies based on case-control or gene candidate approach will be important to identify new variants associated with obesity susceptibility and consequently unveiling its genetic architecture. This will lead to an improvement of our understanding about underlying mechanisms involved in development and origin of the actual obesity epidemic. The integration of several omics will also provide insights about the interplay between genes and environments contributing to the obese phenotype.

Hereditary nonspherocytic hemolytic anemia caused by red cell glucose-6-phosphate isomerase (GPI) deficiency in two Portuguese patients: Clinical features and molecular study.

Glucose-6-phosphate isomerase (GPI) deficiency cause hereditary nonspherocytic hemolytic anemia (HNSHA) of variable severity in individuals homozygous or compound heterozygous for mutations in GPI gene. This work presents clinical features and genotypic results of two patients of Portuguese origin with GPI deficiency. The patients suffer from a mild hemolytic anemia (Hb levels ranging from 10 to 12.7g/mL) associated with macrocytosis, reticulocytosis, hyperbilirubinemia, hyperferritinemia and slight splenomegaly. Genomic DNA sequencing revealed in one patient homozygosity for a new missense mutation in exon 3, c.260G>C (p.Gly87Ala), and in the second patient compound heterozygosity for the same missense mutation (p.Gly87Ala), along with a frameshift mutation resulting from a single nucleotide deletion in exon 14, c.1238delA (p.Gln413Arg fs*24). Mutation p.Gln413Arg fs*24 is the first frameshift null mutation to be described in GPI deficiency. Molecular modeling suggests that the structural change induced by the p.Gly87Ala pathogenic variant has direct impact in the structural arrangement of the region close to the active site of the enzyme.

Polymorphic variations influencing fetal hemoglobin levels: association study in beta-thalassemia carriers and in normal individuals of Portuguese origin.

Three major loci have been associated with HbF levels, including -158C/T (XmnI) at HBG2 promoter region, and several polymorphisms at BCL11A intron-2 and HBS1L-MYB (HMIP) intergenic region. Mutations in the KLF1 gene were recently associated with increased HbF levels. This study aims to evaluate whether genetic variability at these loci influences HbF levels in ß-thalassemia carriers and in normal individuals of Portuguese origin. Sixty five ß-thalassemia carriers, HbF levels ranging from 0.2% to 9.5%, and 60 individuals with normal hematological parameters, HbF levels ranging from 0.2% to 7.4%, were selected for this study. In ß-thal carriers linear regression models revealed a strong statistical significant association for HBG2 (XmnI) rs7482144 (ß=0.455; P=5.858×10(-7)), and nominal significance for BCL11A rs766432 (ß=0.215; P=0.029) and HMIP rs9399137 (ß=0.209; P=0.011). In normal individuals, a case (HbF>2%; n=15) vs. control (HbF<1.7%; n=45) model, showed nominal significant associations for BCL11A SNPs rs11886868 (OR=4; P=0.001), rs766432 (OR=3.7; P=0.002) and rs7606173 (OR=0.36; P=0.032). KLF1 rs3817621 was not found associated with HbF levels. Our results suggest that in Portuguese ß-thal carriers the HBG2 XmnI polymorphism is strongly associated with HbF levels. In normal individuals, BCL11A polymorphisms, but not HMIP or HBG2 (XmnI) loci, are nominally associated with HbF expression.

Familial thrombotic risk based on the genetic background of Protein C Deficiency in a Portuguese Study.

INTRODUCTION: Inherited protein C (PC) deficiency is a well-known risk factor for venous thrombosis (VT). Plasma PC levels are reliable in moderate to severe deficiencies; however, in mildly deficient individuals, the levels may overlap with those considered normal. Genetic studies of PROC, which encodes PC, could help identify carriers; genome-wide association studies (GWAS) have shown that approximately 50% of phenotypic variation in PC deficiency is caused by the cumulative effects of mutations in several other loci, namely in the PROCR. PATIENTS AND METHODS: With the main objective of determining the genotype/phenotype correlation in 59 Portuguese individuals from 26 unrelated families with history of thrombosis and repeatedly low/borderline PC plasma levels, we conducted a molecular study by direct sequencing of PROC; PROC promoter haplotypes and PROCR c.4600A>G polymorphism (rs867186), which are known to influence plasma PC concentrations, were also screened. RESULTS: Twelve different PROC mutations were identified, one of them not previously reported, p.Cys105Arg. The mutation types and locations as well as haplotype combinations correlated with the phenotypic severity. The most frequent mutation, p.Arg199X, correlated with the CGTC haplotype and was identified in nine families containing patients with higher numbers of VT episodes. This mutation in homozygous individuals for the CGTC haplotype is a significant risk factor for VT in Portuguese. CONCLUSION: These genetic family studies allowed the identification of the unknown carriers and individuals at a higher thrombotic risk within each family, thus permitting the evaluation of the need for prophylactic measures, particularly in at-risk situations.

Influence of physical activity on the association between the FTO variant rs9939609 and adiposity in young adults.

OBJECTIVES: To investigate in a population sample of Portuguese young adults the association of the FTO variant rs9939609 with obesity, BMI, and body-fat and interaction with physical activity (PA) on obesity-susceptibility. METHODS: SNP rs9939609 A/T was genotyped in 550 subjects (231 males and 319 females; 18-36 years old; mean age 21 years old) by TaqMan assay. PA was assessed with a validated self-reported questionnaire of IPAQ. RESULTS: We replicated the association of rs9939609-A risk allele with BMI (P = 0.04) and fat-mass (P = 0.031), and with overweight (including obesity) under a recessive model (P = 0.034). Stratified analyses showed (i) a significant association with overweight/obesity in inactive individuals (P = 0.02) but not in a group reporting participation in sports (P = 0.97). Spearman's correlation test suggested that the impact of a successive increase in PA was a decrease in the body-fat percentage (r = -0.16; P = 0.0002), which is accentuated for homozygous AA (r = -0.34; P = 0.002), and an increase in BMI (r = 0.14; P = 0.001), with a statistically significant correlation for homozygous TT (r = 0.22; P = 0.002). CONCLUSIONS: This study reveals interactions between rs9939609 and PA on obesity indices in Portuguese young adults, suggesting a change in the different body components (lean and fat mass) depending on the FTO genotypes.

Novel variants in the MC4R and LEPR genes among severely obese children from the Iberian population.

We screened for mutations in the MC4R and LEPR genes and investigated the genotype-phenotype correlation in obese individuals belonging to families with evident hereditary patterns of severe and early-onset obesity among the Iberian population. A total of 202 unrelated and severely obese patients since childhood, were enrolled in the study. Bidirectional sequencing of the MC4R gene was carried out in all patients; the LEPR gene was sequenced in 15 individuals based on additional clinical signals. Segregation analysis and/or genotype-phenotype description was performed for subjects with the new mutations and with presumably functional variants. Ten MC4R gene mutations were identified in the heterozygous state in 10 patients. Mutations p.R147G and p.G323E are new and mutations p.R7H, p.G32E, p.H76R, p.V103I, p.S127L, p.T150I, p.I251L and p.G252S were previously described. A new dinucleotide insertion -77_-76insTA in the promoter region of the LEPR gene was found in the heterozygous state in one patient. The new p.R147G and the previously published p.R7H, p.S127L, p.T150I and p.G252S MC4R mutations, cosegregate with obesity in our patients and were predicted to be deleterious. For the novel MC4R p.G323E and LEPR -77_-76insTA gene mutations, the genotype-phenotype correlation and bioinformatic analysis did not clarify whether these mutations are indeed implicated in obesity.

Association study of common polymorphisms in MSRA, TFAP2B, MC4R, NRXN3, PPARGC1A, TMEM18, SEC16B, HOXB5 and OLFM4 genes with obesity-related traits among Portuguese children.

At least 52 genetic loci were associated with obesity-related traits. However, little is known about the genetic basis of obesity among children. This study aims to test whether 10 polymorphisms in obesity-related genes methionine sulfoxide reductase A (MSRA), transcription factor AP-2 beta (TFAP2B), melanocortin 4 receptor (MC4R), neurexin 3 (NRXN3), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), transmembrane protein 18 (TMEM18), homolog of S. cerevisiae Sec16 (SEC16B), homeobox B5 (HOXB5) and olfactomedin 4 (OLFM4) are associated with the risk of obesity in Portuguese children. A total of 730 children aging from 6 to 12 years old, recruited randomly from public schools in Portugal, were analysed. Anthropometric measurements were obtained and children were classified into three phenotypic groups, normal weight (n=256), overweight (n=320) and obese (n=154), according to the International Obesity Task Force cutoffs. Polymorphisms were genotyped by allelic discrimination TaqMan assays. The MC4R rs12970134 polymorphism was nominally associated with body mass index (BMI) (P=0.035), BMI Z-score (P=0.043) and waist circumference (P=0.020), and borderline associated with weight (P=0.053). Near nominal associations were also found for the PPARGC1A rs8192678 polymorphism with weight (P=0.061), and for the MSRA rs545854 polymorphism with BMI (P=0.055) and BMI Z-score (P=0.056). Furthermore, logistic regression showed that MC4R rs12970134 and TFAP2B rs987237 were nominally, respectively, associated (P=0.029) and borderline associated (P=0.056) with the obese phenotype. This study highlighted the possible association of MC4R, PPARGC1A, MSRA and TFAP2B polymorphisms with several obesity-related traits in a sample of Portuguese children. The two significant associated TFAP2B rs987237 and MC4R rs12970134 polymorphisms showed an opposite direction of effect to that in the original reports.

Predicting age from blood by droplet digital PCR using a set of three DNA methylation markers.

Evaluation of DNA methylation (DNAm) patterns is a promising tool for age estimation. The duplex droplet digital PCR (ddPCR) method has been recently investigated for DNAm evaluation, revealing to be a potential methodology for DNAm evaluation and molecular age estimation. In this study, we evaluated DNAm levels of CpGs located at the three age-associated genes ELOVL2, FHL2 and PDE4C using ddPCR to develop an age prediction model. Blood-derived DNA samples from 58 healthy individuals (42 women and 16 men; aged 1-93 years old) were submitted to bisulfite conversion followed by ddPCR using dual-labeled probes targeting methylated and unmethylated DNA sequences. Simple linear regression statistics revealed a strong correlation between DNAm levels and chronological age for FHL2 (R = 0.948; P = 1.472 × 10-29) and PDE4C (R = 0.819; P = 3.917 × 10-15), addressing only one CpG for each gene. For the ELOVL2 gene, evaluating five CpG sites in simultaneous, revealed a strong age correlation (R = 0.887; P = 2.099 × 10-20) in a simple linear regression statistics and very strong age correlation (R = 0.926; P = 2.202 × 10-25) when using quadratic regression statistics. The multivariable regression analysis, using methylation information captured on ELOVL2 (squared), FHL2 and PDE4C genes, revealed a very strong age correlation (R = 0.970; P = 5.356 ×10-33), explaining 93.7 % of age variance, displaying a mean absolute deviation (MAD) between chronological and predicted age of 4.657 years (RMSE = 6.044). We postulate that the ddPCR method should be further investigated for DNAm-based age prediction, because it is a relatively simple and an accurate method that can be routinely used in forensic laboratories for testing a few numbers of markers.

The lactase persistence -13910C>T polymorphism shows indication of association with abdominal obesity among Portuguese children.

AIM: The -13910C>T single nucleotide polymorphism located upstream of the lactase gene (LCT) was found tightly associated with lactase persistence in European populations. Recently, it was also associated with body mass index (BMI) and obesity in European adults. The aim of this study was to test the association of -13910C>T polymorphism with obesity-related traits and risk of obesity in children. METHODS: We genotyped 580 Portuguese children (6-12-year-olds) for the -13910C>T polymorphism using TaqMan probes by real-time PCR. Anthropometric measurements were assessed in all children. Obesity was defined according to the International Obesity Task Force (IOTF) cut-offs and abdominal obesity using the sex and age-specific ≥90th waist circumference percentile. RESULTS: We found indication for an association between the-13910*T allele and children abdominal obesity (odds ratio [OR] = 1.41; 95% confidence intervals [CI]: 1.03-1.94; p = 0.030). Under the dominant model, the indicative association was observed between the LCT-13910 CT/TT genotypes and abdominal obesity, remaining significant after adjustment for age and gender (OR = 1.65; 95% CI: 1.04-2.60; p = 0.029). No association was detected with the risk of obesity (p = 0.350). CONCLUSION: Our results suggest that the -13910C>T polymorphism may predispose to abdominal obesity in Portuguese children. The association with BMI or risk of obesity, previously observed in adults, was not confirmed.

Distribution of the - 13910C>T polymorphism in the general population of Portugal and in subjects with gastrointestinal complaints associated with milk consumption.

BACKGROUND: The - 13910C>T polymorphism has been associated with lactase persistence (LP) in European populations. AIM: To assess - 13910C>T genotypes across Portugal and in adult individuals with unspecific gastrointestinal complaints associated with milk consumption. SUBJECTS AND METHODS: This study genotyped - 13910C>T in the general population from Northern (n = 64), Central (n = 70) and Southern (n = 65) Portugal and in 40 subjects with gastrointestinal symptoms. Additionally, the concordance was evaluated between breath-hydrogen test and - 13910C>T genotypes in 65 samples. RESULTS: An overall frequency of 0.349 for the LP - 13910*T allele was estimated in the general population, with a noticeable decrease in the South (0.269) compared with North (0.383) and Centre (0.393). Among the symptomatic group, the frequency of the - 13910*T allele (0.363) was not significantly different from the general population. A 94% concordance was found between the breath-hydrogen and the molecular tests. CONCLUSIONS: This study suggests that (i) the distribution of the LP polymorphism is not uniform across the country, (ii) genotyping - 13910C>T is a good diagnostic tool for lactase status in the Portuguese population and (iii) self-reported gastrointestinal complaints are not good predictors of the LP status, implying that a significant part of those complaints may not be related to hypolactasia.