Challenges and (Un)Certainties for DNAm Age Estimation in Future

Age estimation is a paramount issue in criminal, anthropological, and forensic research. Because of this, several areas of research have focused on the establishment of new approaches for age prediction, including bimolecular and anthropological methods. In recent years, DNA methylation (DNAm) has arisen as one of the hottest topics in the field. Many studies have developed age- prediction models (APMs) based on evaluation of DNAm levels of many genes in different tissue types and using different methodological approaches. However, several challenges and confounder factors should be considered before using methylation levels for age estimation in forensic contexts. To provide in-depth knowledge about DNAm age estimation (DNAm age) and to understand why it is not yet a current tool in forensic laboratories, this review encompasses the literature for the most relevant scientific works published from 2015 to 2021 to address the challenges and future directions in the field. More than 60 papers were considered focusing essentially on studies that developed models for age prediction in several sample typesinfo:eu-repo/semantics/publishedVersio

A Blood–bone–tooth model for age prediction in forensic contexts

The development of age prediction models (APMs) focusing on DNA methylation (DNAm) levels has revolutionized the forensic age estimation field. Meanwhile, the predictive ability of multi-tissue models with similar high accuracy needs to be explored. This study aimed to build multi-tissue APMs combining blood, bones and tooth samples, herein named blood–bone–tooth-APM (BBT-APM), using two different methodologies. A total of 185 and 168 bisulfite-converted DNA samples previously addressed by Sanger sequencing and SNaPshot methodologies, respectively, were considered for this study. The relationship between DNAm and age was assessed using simple and multiple linear regression models. Through the Sanger sequencing methodology, we built a BBT-APM with seven CpGs in genes ELOVL2, EDARADD, PDE4C, FHL2 and C1orf132, allowing us to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 6.06 years, explaining 87.8% of the variation in age. Using the SNaPshot assay, we developed a BBT-APM with three CpGs at ELOVL2, KLF14 and C1orf132 genes with a MAD of 6.49 years, explaining 84.7% of the variation in age. Our results showed the usefulness of DNAm age in forensic contexts and brought new insights into the development of multi-tissue APMs applied to blood, bone and teethinfo:eu-repo/semantics/publishedVersio

Analysis of malaria associated genetic traits in Cabo Verde, a melting pot of European and sub Saharan settlers

Malaria has occurred in the Cabo Verde archipelago with epidemic characteristics since its colonization. Nowadays, it occurs in Santiago Island alone and though prophylaxis is not recommended by the World Health Organization, studies have highlight the prospect of malaria becoming a serious public health problem as a result of the presence of antimalarial drug resistance associated with mutations in the parasite populations and underscore the need for tighter surveillance. Despite the presumptive weak immune status of the population, severe symptoms of malaria are not observed and many people present a subclinical course of the disease. No data on the prevalence of sicklecell trait and red cell glucose-6-phosphate dehydrogenase deficiency (two classical genetic factors associated with resistance to severe malaria) were available for the Cabo Verde archipelago and, therefore, we studied the low morbidity from malaria in relation to the particular genetic characteristics of the human host population. We also included the analysis of the pyruvate kinase deficiency associated gene, reported as putatively associated with resistance to the disease. Allelic frequencies of the polymorphisms examined are closer to European than to African populations and no malaria selection signatures were found. No association was found between the analyzed human factors and infection but one result is of high interest: a linkage disequilibrium test revealed an association of distant loci in the PKLR gene and adjacent regions, only in non-infected individuals. This could mean a more conserved gene region selected in association to protection against the infection and/or the disease

Pyruvate-Kinase Deficiency in the Portuguese Population - A Molecular and Population Study

Tese de doutoramento em Antropologia (Antropologia Biológica) apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraO estudo molecular dos doentes de naturalidade Portuguesa com anemia hemolítica por deficiência de piruvato-quinase (PK; EC 2.7.1.40) permitiu identificar 8 mutações diferentes no gene PKLR. Seis mutações são descritas pela primeira vez: 3 mutações missense [1435C>T(479Arg>Cys), 1670G>C(557Gly>Ala) e 1706G>A(569Arg>Gln)]; 2 mutações de splicing [IVS8(+2)T>G e IVS10(+1)G>C]; e a substituição –72A>G na região promotora. Foram ainda encontradas duas mutações missense [1456C>T(486Arg>Trp) e 993C>A(331Asp>Glu)], previamente descritas. As mutações missense resultam na troca de aminoácidos em zonas conservadas da subunidade PK-R, reduzindo a eficiência catalítica da enzima. A mutação 1435C>T ocorre no último dinucleótido do exão 10 e deverá afectar também o processo de splicing. A mutação -72A>G ocorre na sequência GATA mais próxima do exão 1, na região promotora R do gene PKLR, e é responsável por uma diminuição drástica da síntese de mRNA. A mutação IVS8(+2)T>G anula o local de splicing 5’ do intrão 8 e, devido à activação de um local alternativo de splicing, leva à inserção dos primeiros 20 nucleótidos do intrão 8 no mRNA. A mutação IVS10(+1)G>C anula o local de splicing 5’ no intrão 10, e leva à não inclusão do exão 10 no mRNA. Foram estudados 5 polimorfismos do gene PKLR [-148C/ T, 1705A/C, 1738C/T, T10/19 e (ATT)n] nos doentes e em 2 amostras populacionais distintas da região Centro de Portugal e de S. Tomé e Príncipe. A associação das mutações com os haplótipos definidos pelos polimorfismos, sugere uma origem única para qualquer uma das mutações recorrentes identificadas. A análise dos marcadores polimórficos nas amostras populacionais de Portugal e de S. Tomé e Príncipe mostra que a população subsariana apresenta níveis mais elevados de heterozigotia, maior diversidade haplotípica e um desequilíbrio gamético menorWe have studied the PKLR gene in unrelated Portuguese patients with haemolytic anaemia associated with erythrocyte Pyruvate Kinase (PK; EC 2.7.1.40) deficiency. Eight different mutations were identified, six of them for the first time: three missense mutations [1435G>A(479Arg>Cys), 1670G>C(557Gly>Ala), 1706G>A(569Arg>Gln)], two 5’ splice site mutations [IVS8(+2)T>G and IVS10(+1)G>C] and the point mutation -72A>G on R-PK promoter. Two previously described missense mutations, 1456C>T(486Arg >Trp) and 993C>A(331Asp>Glu), were also found. All missense mutations cause aminoacids substitutions in conserved regions of the PK subunit, affecting the catalytic efficiency of the enzyme. The new mutation 1435C>T in the last dinucleotide of exon 10 probably also affects the normal splicing of intron 10. The new regulatory mutation -72A>G within the GATA-A element in the R-type promoter region severely decreases the PK mRNA synthesis. Mutation IVS8(+2)T>G leads to a 20 nucleotides longer spliced transcript, due to the abolishment of the 5’ donor splice site and the activation of a cryptic splice site in intron 8. The IVS10(+1)G>C mutation, abolishing the intron 10 donor splice site, leads to the skipping of exon 10 in the mRNA transcript. Five intragenic PKLR polymorphisms [-148C/T, 1705A/C, 1738C/T, T10/19 and (ATT)n] were studied in PK-deficient patients and in two normal population samples of Central Portugal and S. Tomé e Príncipe. The haplotype analysis with the PKLR mutations using these five polymorphic markers suggests a single origin for each of the recurrent mutations found in the Portuguese population. The analysis of the polymorphic PKLR markers in the two distinct population samples of Central Portugal and S. Tomé e Príncipe showed higher heterozygosity values in the sub-saharan population, together with the presence of more haplotypes and lower linkage disequilibrium

Estudo populacional de 6 STRs (TH01, TPOX, CSF1PO, D7S820, D13S317 e D16S539) na região Centro de Portugal

As frequências alélicas de 6 STRs autossómicos (TH01, TPOX, CSF1PO, D7S820, D13S317 e D16S539), correntemente utilizados em estudos forenses, foram estimadas numa amostra de indivíduos não aparentados naturais da região Centro de Portugal (N=70-104). Não se verificaram desvios significativos ao equilíbrio de Hardy-Weinberg para os marcadores estudados. Os resultados foram comparados com os obtidos em estudos realizados noutras regiões do país (Norte, Centro e Sul de Portugal, Madeira e Açores) e em diversas populações Europeias não tendo sido encontradas diferenças estatisticamente significativas para a maioria dos loci, o que mostra, por um lado, semelhança genética da região Centro de Portugal com estas populações e, por outro, a consistência dos resultados encontrados

Association study between near-MC4R variants and obesity-related variables in Portuguese young adults

The aim of this study was to investigate in a population sample of Portuguese young adults i) the association of near-MC4R variants with obesity measures; and ii) the presence of mutations in MC4R coding region contributing to severe obesity. Polymorphisms rs17782313, rs12970134 and rs9944545 were genotyped in 544 subjects (225 males, 319 females; mean age 20.7 years) by TaqMan assay. Body Mass Index (BMI) was calculated (kg/m2) and percentage of body fat (%FAT) was measured using bioimpedance analysis. The coding region of MC4R gene was examined in nine severe obese subjects (BMI > 35 kg/m2) by Sanger sequencing. Linear regression showed no associations between variants rs17782313, rs12970134 and rs9944545 and BMI (P = 0.459, P = 0.691 and P = 0.611, respectively) or %FAT (P = 0.853, P = 0.678 and P = 0.434, respectively). The logistic regression, in the additive model, revealed no statistically significant interactions with overweight/obesity for any SNP (P = 0.717, P = 0.771 and P = 0.417, respectively). Also, haplotype analysis revealed no significant associations testing for BMI, %FAT or the obesity status. Genomic DNA sequencing of the MC4R coding region revealed no variations explaining the severely obese phenotype. As main conclusion, this study in young Portuguese adults does not replicate previous findings evidencing the association of near-MC4R polymorphisms with human obesity. Nevertheless, the obtained data is in accordance with several studies reporting no such associations

Association of FTO Polymorphisms with Obesity and Obesity-Related Outcomes in Portuguese Children

Several studies have reported an association between single nucleotide polymorphisms in the first intron of the FTO gene and body mass index (BMI) or obesity. However, this association has not yet been studied among the Portuguese population. This study aims to assess the association of three FTO polymorphisms (rs1861868, rs1421085 and rs9939609) with obesity-related outcomes in a sample of Portuguese children.We examined a total of 730 children, 256 normal-weight (55.9% girls), 320 overweight (45.3% girls) and 154 obese (53.2% girls), aging from 6 to 12-years-old, recruited randomly from public schools in the central region of Portugal. DNA samples were genotyped for the three polymorphisms by allelic discrimination TaqMan assay. Association of the FTO polymorphisms with several anthropometric traits was investigated. Additionally, we tested association with the risk of obesity using overweight and obese vs. normal-weight children.We found significant associations of rs9939609 and rs1421085 polymorphisms with weight, BMI, BMI Z-score, waist circumference and hip circumference, even after age and gender adjustment (p<0.05 in all traits). For rs1861868 polymorphism, marginally significant associations were obtained with weight (p = 0.081) and BMI (p = 0.096) after adjustment for age and gender. In case-control studies, both rs9939609 and rs1421085 polymorphisms were significantly associated with obesity (OR 1.97; 95% CI, 1.08-3.59; p = 0.026; OR 2.11; 95% CI, 1.17-3.81; p = 0.013, respectively) but not with overweight (p>0.05). Haplotype analyses identified two combinations (ACA and GCA) associated with a higher risk of obesity (OR 1.53; 95% CI, 1.06-2.22; p = 0.023; OR 1.73; 95% CI, 1.06-2.87; p = 0.030, respectively).This study provides the first evidence for the association of FTO polymorphisms with anthropometric traits and risk of obesity in Portuguese children

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

Association study between near-MC4R variants and obesity-related variables in Portuguese young adults

The aim of this study was to investigate in a population sample of Portuguese young adults i) the association of near-MC4R variants with obesity measures; and ii) the presence of mutations in MC4R coding region contributing to severe obesity. Polymorphisms rs17782313, rs12970134 and rs9944545 were genotyped in 544 subjects (225 males, 319 females; mean age 20.7 years) by TaqMan assay. Body Mass Index (BMI) was calculated (kg/m2) and percentage of body fat (%FAT) was measured using bioimpedance analysis. The coding region of MC4R gene was examined in nine severe obese subjects (BMI > 35 kg/m2) by Sanger sequencing. Linear regression showed no associations between variants rs17782313, rs12970134 and rs9944545 and BMI (P = 0.459, P = 0.691 and P = 0.611, respectively) or %FAT (P = 0.853, P = 0.678 and P = 0.434, respectively). The logistic regression, in the additive model, revealed no statistically significant interactions with overweight/obesity for any SNP (P = 0.717, P = 0.771 and P = 0.417, respectively). Also, haplotype analysis revealed no significant associations testing for BMI, %FAT or the obesity status. Genomic DNA sequencing of the MC4R coding region revealed no variations explaining the severely obese phenotype. As main conclusion, this study in young Portuguese adults does not replicate previous findings evidencing the association of near-MC4R polymorphisms with human obesity. Nevertheless, the obtained data is in accordance with several studies reporting no such associations