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Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

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Induced pluripotent stem cell (iPSC) lines were generated from erythroblasts of two patients with amyotrophic lateral sclerosis (ALS) and two healthy individuals. One familial and one sporadic ALS patients were used, both with genetic alterations in VAPB gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the iPSC cell lines. The four iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

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OBJECTIVE: We investigated whether calcium plus vitamin D supplementation interacts with polymorphisms in the VDR gene promoter region to affect changes on maternal bone mass from 5 to 20 wk postpartum in Brazilian adolescent mothers. METHODS: Pregnant adolescents (14-19 y) randomly received calcium plus cholecalciferol (600 mg/d + 200 IU/d, n = 30) or placebo (n = 26) from 26 wk of pregnancy until parturition. Bone mineral content (BMC), bone area (BA), and bone mineral density (BMD) at total body, lumbar spine, total hip, and femoral neck were evaluated at 5 and 20 wk postpartum. Serum 25-hydroxyvitamin D (25[OH]D) and parathyroid hormone concentrations were measured. Real-time polymerase chain reaction was used for genotyping rs7139166 (1521 pb G > C) and rs4516035 (1012 pb A > G). Interactions between supplementation and polymorphisms were adjusted for significant covariates. RESULTS: Changes in serum 25(OH)D from pregnancy to postpartum differed between supplemented and placebo groups for mothers carrying 1521 GG/1012 AA genotypes (P = 0.004). Only in the placebo group, mothers carrying 1521 GG/1012 AA had greater reduction in total BMD z score, femoral neck BMC, and BMD from 5 to 20 wk postpartum compared with those with 1521 GC/1012 AG (P < 0.05). In the placebo group, total hip BA decreased from 5 to 20 wk postpartum in adolescents with 1521 GG/1012 AA, but increased in those with 1521 GC/1012 AG (P < 0.05), in contrast to the supplemented group. CONCLUSION: Calcium plus vitamin D supplementation during pregnancy interacted with polymorphisms in the VDR gene promoter region affecting postpartum bone loss. The increased supply of calcium and vitamin D appeared to minimize postpartum bone loss particularly in adolescents with 1521 GG/1012 AA.

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Atherosclerosis is a complex disease, involving both genetic and environmental factors. However, the influence of genetic variations on its early development remains unclear. This study examined the association of 12 different polymorphisms with atherosclerosis severity in anterior descending coronary (DA, n = 103) and carotid arteries (CA, n = 66) of autopsied young adults (< 30 years old). Histological sections (H-E) were classified according to the American Heart Association. Polymorphisms in ACE, TNF-α (- 308G/A and - 238 G/A), IFN-γ (+ 874 A/T), MMP-9 (- 1562 C/T), IL-10 (- 1082 A/G and - 819 C/T), NOS3 (894 G/T), ApoA1 (rs964184), ApoE (E2E3E4 isoforms), and TGF-ß (codons 25 and 10) genes were genotyped by gel electrophoresis or automatic DNA sequencing. Firearm projectile or car accident was the main cause of death, and no information about classical risk factors was available. Histological analysis showed high prevalence of type III atherosclerotic lesions in both DA (69%) and CA (39%) arteries, while severe type IV and V lesions were observed in 14% (DA) and 33% (CA). Allele frequencies and genotype distributions were determined. Among the polymorphisms studied, IFN-γ and IL-10 (- 1082 A/G) were related to atherosclerosis severity in DA artery. No association between genotypes and lesion severity was found in CA. In conclusion, we observed that the high prevalence of early atherosclerosis in young adults is associated with IFN-γ (p < 0.001) and IL-10 (p = 0.013) genotypes. This association is blood vessel dependent. Our findings suggest that the vascular system presents site specialization, and specific genetic variations may provide future biomarkers for early disease identification.

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UNLABELLED: Retinoblastoma (RB) is a malignant neoplasia that occurs mostly in children under 5 years. Recently, CDKN1A gene has been shown to be up-regulated in a context of loss of function of pRb. This gene encodes the p21 protein, which is the bona fide effector of p53. We hypothesized whether two putatively functional single nucleotide polymorphisms (SNPs) of CDKN1A (rs1801270 C>A and rs1059234 C>T) may influence the risk and/or survival of RB patients. We genotyped both SNPs in 141 RB patients and 120 unrelated healthy individuals. Statistical analyses consisted of chi-square (χ(2)), odds ratio (OR) and survival curves by Kaplan-Meier method. We found that patients who carry the genotype CA for rs1801270 and CT for rs1059234 were associated to an increased risk of RB [OR = 2.5, 95% confidence interval (CI) = 1.38-4.53], whereas patients with CC for both polymorphisms were associated to a lower risk of developing RB (OR = 0.43, 95% CI = 0.25-0.74). On the other hand, Kaplan-Meier curves did not show statistically significant differences in survival among the studied polymorphisms. We conclude that the minor alleles of rs1801270 and rs1059234 polymorphisms may act as risk factors for the development of RB in our sample. SUMMARY: The minor alleles of polymorphisms rs1801270 C>A and rs1059234 C>T in CDKN1A (p21) gene may act as risk factors for the development of RB; however, they do not seem to influence overall survival.

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N-Acetyltransferase 2 (NAT2) metabolizes a variety of xenobiotics that includes many drugs, chemicals and carcinogens. This enzyme is genetically variable in human populations and polymorphisms in the NAT2 gene have been associated with drug toxicity and efficacy as well as cancer susceptibility. Here, we have focused on the identification of NAT2 variants in Brazilian individuals from two different regions, Rio de Janeiro and Goiás, by direct sequencing, and on the characterization of new haplotypes after cloning and re-sequencing. Upon analysis of DNA samples from 404 individuals, six new SNPs (c.29T>C, c.152G>T, c.203G>A, c.228C>T, c.458C>T and c.600A>G) and seven new NAT2 alleles were identified with different frequencies in Rio de Janeiro and Goiás. All new SNPs were found as singletons (observed only once in 808 genes) and were confirmed by three independent technical replicates. Molecular modeling and structural analysis suggested that p.Gly51Val variant may have an important effect on substrate recognition by NAT2. We also observed that amino acid change p.Cys68Tyr would affect acetylating activity due to the resulting geometric restrictions and incompatibility of the functional group in the Tyr side chain with the admitted chemical mechanism for catalysis by NATs. Moreover, other variants, such like p.Thr153Ile, p.Thr193Met, p.Pro228Leu and p.Val280Met, may lead to the presence of hydrophobic residues on NAT2 surface involved in protein aggregation and/or targeted degradation. Finally, the new alleles NAT2*6H and NAT2*5N, which showed the highest frequency in the Brazilian populations considered in this study, may code for a slow activity. Functional studies are needed to clarify the mechanisms by which new SNPs interfere with acetylation.

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The genetic influence on bone mineralization during adolescence is unclear possibly due to modifying factors such as skeletal maturation and lifestyle. We evaluated the influence of polymorphisms of the vitamin D receptor (VDR) gene on longitudinal changes in bone mass, bone- and calcium-related hormones in 46 adolescent soccer players (11.8-14.2 years). Total body bone mineral content (TBMC) and density (TBMD) were measured at baseline and after 6 months. Insulin-like growth factor-I (IGF-1), testosterone, intact parathyroid hormone, and activity of plasma bone alkaline phosphatase were measured at baseline and after 3 months. The influence of FokI or TaqI VDR genotypes on changes in the outcome variables were analyzed by univariate ANOVA with adjustment for chronological age, skeletal age and body weight at baseline. At baseline, boys with Ff genotype had higher TBMC, TBMD, TBMD Z-score compared to those with FF genotype (P < 0.05). After 3 months, Ff boys also had higher increment in plasma IGF-1 (P < 0.05). FokI polymorphism did not influence changes in bone mass measurements after 6 months, although differences detected at baseline remained significant after 6 months. There were no differences in the outcome variables according to TaqI genotypes. This study demonstrates that FokI polymorphisms affect bone mass in Brazilian adolescent soccer players and suggests that the FokI effect on bone mineralization occurs during bone maturation, possibly at the initial pubertal stages.

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Clitoromegaly in the neonatal period is an important morphologic sign that can be useful for sexual determination in aberrant cases. In rhesus monkeys, differentiation of the external genitalia occurs early during gestation (at 55 to 60 d) and is complete by approximately 80 d. Most of the critical steps in genital differentiation in primates occur prenatally. We sought to determine clitoral size in normal rhesus monkeys (Macaca mulatta) and possible effects of age and inheritance. Clitoral length was highly variable and had no relationship to fertility. Statistical evaluation revealed no association in the distribution of daughters with and without clitoris between mothers with and without clitoris. However, even when mated with several female monkeys, some male macaques produced primarily daughters without clitoris.

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BACKGROUND: Whether insulin resistance and not obesity per se is the major contributor to clinical outcomes associated with obesity has not been fully established. This study evaluated in a group of obese Brazilians of multiethnic origin to what extent the prevalence of hypertension and other cardiometabolic risk factors varies as a function of the degree of insulin sensitivity. METHODS: The study involved 118 individuals (mean age of 44+/-12 years; BMI=38.6+/-7.9 kg/m(2)) without evidence of diabetes or cardiovascular disease. Insulin resistance was assessed by HOMA-IR index, which was used to stratify patients into tertiles. RESULTS: The mean HOMA-IR in tertile 1, the most insulin-sensitive group, was 2.7+/-0.8 and in tertile 3, the most insulin-resistant group, 9.1+/-2.4 (P<0.001). Mean arterial pressure showed a linear and significant variation across the HOMA-IR tertiles 1, 2, and 3 (94.3+/-11.7; 98.7+/-11.4; 105.0+/-12.4 mm Hg), as did fasting plasma glucose (93.6+/-12.1; 98.1+/-12.7; 100.0+/-11.0 mg/dL), uric acid (4.7+/-1.4; 5.9+/-1.9; 6.3+/-1.4 mg/dL), HDL-cholesterol (48.1+/-11.6; 46.5+/-10.5; 42.2+/-8.0 mg/dL), and plasma adiponectin (7.8+/-3.3; 7.0+/-2.8; 6.3+/-6.5 microg/mL), respectively. The results indicated that 27.5% of our patients had dysglicemia, 28.2% had hypertriglyceridemia, and 30.7% had arterial hypertension in the most insulin-sensitive tertile, when compared with 51%, 53.8% and 79.4%, respectively, in the most insulin-resistant tertile. A stepwise regression analysis showed that only HOMA-IR and age independently affected the risk for increased systolic blood pressure. CONCLUSION: In conclusion, our findings have shown that the risk of developing essential hypertension, type 2 diabetes, and cardiovascular disease is accentuated in obese individuals who are also more insulin resistant.

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BACKGROUND: This study assessed in obese Brazilians subjects whether a common variant of leptin gene, -2548G>A, is associated with blood pressure changes. METHODS: A total of 140 subjects, 99 women; mean age of 45.2 +/- 12.4 years; body mass index (BMI) = 38.5 +/- 8.0 kg/m2 were included. Blood pressure was recorded using Dinamap 1846 (Critikon, Tampa, FL). Molecular analysis was made by use of PCR and restriction fragment-length polymorphism analysis. Plasma insulin and leptin concentrations were determined by radioimmunoassay. RESULTS: AA homozygotes, in comparison with the G-allele carriers, showed significant lower levels of systolic, diastolic, and mean arterial pressure (120 +/- 10 vs. 132 +/- 17 mm Hg, P = 0.01; 75 +/- 6 vs. 84 +/- 12 mm Hg, P = 0.009; 92 +/- 7 vs. 100 +/- 12 mm Hg, P = 0.007, respectively). The differences in blood pressure remained significant after adjusting for the influence of gender, age, obesity, and body fat distribution as well as for leptin, insulin, and homeostasis model assessment of insulin resistance. A stepwise regression analysis confirmed that the LEP AA genotype independently predicted blood pressure changes. On the other hand, in GG homozygotes, insulinemia showed a significant association with blood pressure values. This suggests that common LEP genotype carriers exhibiting high insulin levels, reflecting an insulin-resistant state, were particularly prone to higher blood pressure levels. CONCLUSIONS: Our results showing that higher blood pressure levels were found with the most prevalent -2548G>A genotype, whereas patients with the AA genotype seemed to be protected from hypertension, indicate that the -2548G>A polymorphism of LEP appears to be an important mediator of obesity hypertension.

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