Uniparental disomy of chromosome 21: A statistical approach and application in paternity tests.

Considering the overall frequency of paternity investigation cases including mutational events, there is a real possibility that at least a fraction of all inconsistencies reported in paternity cases are caused not by polymerase slippage mutations, but to chromosomic abnormalities, as Uniparental Disomy (UPD). We report here the investigation of a trio paternity case (mother, child and alleged father), with observed inconsistencies that can alternatively be explained by occurrence of maternal uniparental isodisomy of chromosome 21 (miUPD21). A total of 350 short tandem repeat (STR) and single nucleotide polymorphism (SNP) markers were tested, statistically suggesting true biological linkage within the trio. Additionally, we propose miUPD21 explains, with significantly greater probability, the occurrence of detected inconsistencies, when compared to alternative hypothesis of multiple and simultaneous slippage mutations. Similar cases could have their statistical conclusions improved or even altered by including unusual chromosomal segregation patterns in the hypothesis formulation, as well as in mathematical calculations. Such reports of allelic inconsistencies being explained by chromosomal alterations are common in clinical genetics, and such situations might have impact on forensic investigation.

Brazilian forensic casework analysis through MPS applications: Statistical weight-of-evidence and biological nature of criminal samples as an influence factor in quality metrics.

Real forensic casework biological evidence can be found in a myriad of different conditions and presenting very distinct features, including key elements such as degradation levels, the nature of biological evidence, mixture presence, and surface or substrate deposition, among others. Technical protocols employed by forensic DNA analysts must consider such characteristics in order to improve the chances of successfully genotyping these materials. MPS has been used as a very useful tool for forensic sample processing and genetic profile generation. However, it is not completely clear how different features encountered with real forensic samples impact sequencing quality and, consequently, profile accuracy and reliability. In this context, the present study analyzes a set of 47 real forensic casework samples, obtained from semen, saliva, blood and epithelial evidence, as well as reference oral swabs, aiming to evaluate the impact of a sample's biological nature in profiling success. All DNA extracts from samples were standardized according to sample conditions, as assessed by traditional forensic profiling methods (real-time PCR quantitation and capillary electrophoresis-coupled STR fragment analysis). Samples were separated into groups according to their biological nature, and the resultant sequencing quality was evaluated through a series of well-established statistical tests, applied specifically to six different MPS quality metrics. The results showed that certain groups of samples, especially epithelial and (to a lesser extent) saliva samples, exhibited significantly lower quality in terms of some of the evaluated metrics. A number of reasons for such unexpected behavior are discussed. In addition, a series of calculations was performed to assess the weight of genetic evidence in Brazilian samples, and reflexes in data analysis and national allele frequency database construction are discussed. Overall, the results indicate that a unified national allele frequency database can be used nationwide. Besides this, MPS genetic profiles obtained from samples with particular biological origins may benefit from meticulous manual review, and visual inspection could be important as an additional step to avoid genotyping errors or misinterpretation, leading to more trustworthy and reliable results in real criminal forensic casework analysis.

Full mtDNA genome sequencing of Brazilian admixed populations: A forensic-focused evaluation of a MPS application as an alternative to Sanger sequencing methods.

The use of Massive Parallel Sequencing (MPS) techniques have been proposed by the forensic community as an alternative to Sanger sequencing methods in routine forensic casework analysis regarding mitochondrial DNA (mtDNA). Interesting features of MPS include high throughput, ability to simultaneously genotype a significant number of samples by barcoding techniques, processing automation, reduced time and costs, among others. Advantages include the capability of generating full mtDNA genome sequences versus usual techniques, usually limited to hypervariable or control regions exclusively. In this work, 96 reference single-source samples from three different Brazilian cities were subjected to full mtDNA genome sequencing by MPS techniques using an early-access version of Precision ID mtDNA Whole Genome Panel on an Ion Torrent PGM platform (Thermo Fisher Scientific, Waltham, MA, USA). Complete, high-quality sequences were obtained and sequencing performance was evaluated via four different metrics. As a subset of evaluated samples have been previously submitted for Sanger sequencing of the control region, a comparative analysis of both methods' results was conducted in order to compare technique adequacy within a forensic context. Even though this study is one of the first to report full mtDNA genome sequences for Brazilian admixed populations, the observed haplotypes exhibit a predominance of Native American and African maternal lineages in the studied sample set, reproducing results described in the literature for control regions only. Interpopulation analysis among Brazilian and 26 worldwide populations was also carried out. The results indicate that MPS-generated full mtDNA genome sequences may have great utility in forensic real casework applications, with a pronounced gain of genetic information and discrimination power provided by coding region evaluation and the enhanced capacity of heteroplasmies determination. Database construction and other relevant factors concerning implementation of such techniques in Brazilian forensic laboratories are also discussed.

APOA5 polymorphisms associated with lipid metabolism in Brazilian children and adolescents.

Single nucleotide polymorphisms in the APOA5 gene have been studied for their association with metabolic syndrome. Thus, elucidating the effect of the mechanism involved in APOA5 gene polymorphisms on lipid metabolism is of great importance. In this study we aimed to determine the allelic and genotypic frequencies of -1131T>C, Ser19Trp, and intergenic APOA4/A5 and to evaluate the association between these variants with plasma lipid levels in children and adolescents from Brazil. This study included 524 healthy children and adolescents from Mother and Child Hospital in Recife, Pernambuco, Brazil. Data were obtained on medical history, drug intake, lifestyle variables, and demography. DNA from collected samples was extracted and genotyped for the three polymorphisms. In this studied population, triglycerides and very low-density protein levels were significantly high in subjects carrying the 19WW genotype (P < 0.001), demonstrating the presence of this genetic risk factor in children and adolescents.

Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction.

Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.

More severe clinical course of cardiovascular dysfunction in intensive care unit patients with the 894TT eNOS genotype.

The endothelial nitric oxide synthase (eNOS) plays an important homeostatic role in the cardiovascular system (CVS) by maintaining appropriate blood pressure through production of nitric oxide. The 894TT genotype of 894G>T (Glu298Asp, rs1799983), a polymorphic variant of eNOS, has been associated with several vascular diseases. On the basis of this strong relationship, we monitored daily 585 critically ill adult patients according to their degree of CVS dysfunction and investigated their disease progression by the 894G>T genotype. To obtain information of the general population, we obtained the 894G>T genotypic and allelic frequencies in a random group of 149 healthy subjects. The patients were genotyped for the eNOS 894G>T polymorphism and daily evaluated according to their degree of CVS dysfunction through the Cardiovascular Sequential Organ Failure Assessment (SOFA) score. The mean value of the global CVS dysfunction score was significantly higher in 894TT patients (1.35 ± 0.57) than in non-894TT patients (1.23 ± 0.37; P = 0.035). This score remained significantly higher in 894TT patients, even in different patient clusters (all patients, septic, and non-septic patients) during the 1st week at the intensive care unit (1.86 ± 0.8 versus 1.63 ± 0.62, P = 0.005; 2.32 ± 0.10 versus 2.06 ± 0.08, P = 0.009; 0.84 ± 0.09 versus 0.64 ± 0.08, P = 0.027; respectively). This result shows that the mean values of the cardiovascular SOFA score were higher in 894TT patients in all subgroups. The present study provides evidence that the 894TT eNOS genotype is associated with a higher degree of CVS dysfunction in critically ill patients.

Influence of the 48867A>C (Asp358Ala) IL6R polymorphism on response to a lifestyle modification intervention in individuals with metabolic syndrome.

We evaluated the response of individuals with metabolic syndrome to lifestyle modification intervention and examined the influence of the 48867A>C (Asp358Ala) IL6R (rs2228145) polymorphism on this response. Participants were randomly divided into two groups: NI, nutritional intervention; NIE, nutritional intervention and exercise practice. Intervention lasted three months and participants completed a comprehensive evaluation and had blood collected for biochemical measurements. Eighty-two sedentary individuals with at least three criteria for metabolic syndrome were included. Comparing metabolic syndrome parameters before and after intervention, a reduction of waist circumference was observed, although significant only for AA and AC genotypes. Also, a decrease in triglyceride levels was observed (significant for AA genotype individuals; for the AC genotype, only in the NIE group). Significant reduction of fasting glucose level was observed in all AA genotype individuals; for the AC genotype, only in the NI group. Systolic blood pressure showed significant reduction in AA and AC genotype individuals. After three months of lifestyle modification intervention, improvement in some of the metabolic syndrome parameters was observed, some associated with the IL6R genotype. At enrollment, participants with genotypes AA and AC showed more severe conditions regarding metabolic syndrome inclusion criteria, supporting previous reports that the A allele is a genetic risk factor. These individuals, however, had a better response to intervention compared to individuals with the CC genotype, suggesting that nutritional control and exercise practice could prevent risks associated with metabolic syndrome more efficiently in individuals bearing the A allele.

The synergy of -260T T CD14 and -308GG TNF-α genotypes in survival of critically ill patients.

Literature suggests that the analysis of several polymorphic genetic markers is more informative than the analysis of a single polymorphism. In this study, we tested whether the shared inheritance of TLR2 and TLR4 and TNF-α allelic variants may act in synergy with -260C>T CD14 SNP on the outcome from critical conditions. We monitored 524 critically ill patients from South Brazilian, daily from the ICU admission to their discharge from hospital, or death. Our results revealed that TLR2, TLR4 or TNF-α SNPs alone did not show a significant role in the outcome from critical illness. However, when we performed a combined analysis with the CD14 inheritance, we detected a significant higher survivor rate in -260TT CD14/-308GG TNF-α individuals (P = 0.037). In the adjusted analysis including the main clinical predictors to mortality, we observed that -260TT/-308GG double-genotype was a significant protective factor towards survival (P = 0.046). An increased probability for survival of -260TT/-308GG was also observed by 'pathway genetic load' analysis (unweighted: P = 0.041; weighted: P = 0.036). When we applied a hazard function analysis with the -260TT/-308GG variable as a discriminating factor, -260TT/-308GG patients group had, in fact, a higher survivor rate (P = 0.024). Connected to the beneficial effect of -260TT CD14, the -308GG TNF-α genotype was protective against the reported over expression of TNF-α caused by -308A rare allele. Results support the hypothesis that the interaction between -260C>T CD14 and -308G>A TNF-α functional SNPs may be synergistically influencing the outcome of critically ill patients.

Dietary fat and apolipoprotein genotypes modulate plasma lipoprotein levels in Brazilian elderly women.

Studies show that genetic polymorphisms in apolipoproteins, which are in charge of lipid transport, predispose to atherogenic dyslipidemia. This study aimed to investigate the impact of apolipoprotein E, A5, and B genotypes and dietary intake on lipid profile in a sample of elderly women in Brazil. Two hundred and fifty-two women (60 years or older) living in the outskirts of the Brazilian Federal District underwent clinical and laboratory assessments to characterize glycemic and lipidemic variables, and also to exclude confounding factors (smoking, drinking, hormone replacement, cognitive impairment, physical activity). Three-day food records were used to determine usual dietary intake, whereas genotypic evaluations were in accordance to established methodologies. Genotype frequencies were consistent with the Hardy-Weinberg equilibrium. Prior to adjustment, individuals carrying the epsilon2 allele showed higher serum levels of triglycerides (P<0.05) and VLDL (P<0.005) compared to epsilon4 carriers, whereas LDL levels were considerably elevated in epsilon4 compared to epsilon2 carriers. In the presence of high intake of total fat or a low ratio of polyunsaturated to saturated fatty acid, epsilon4 carriers lost protection against hypertriglyceridemia. There was no association of the apolipoprotein A5 and B genotypes with lipidemic levels independently of the fat intake regimen. Results are suggestive of a dysbetalipoproteinemic-like phenotype in postmenopausal women, with remarkable gene-diet interaction.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina.

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8%; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2%; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95% CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8 percent; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2 percent; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95 percent CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.

Low activity of mitochondrial HMG-CoA synthase in liver of starved piglets is due to low levels of protein despite high mRNA levels.

The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65-73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria.

Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet.

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.