Generation of patient-specific pluripotent induced stem cell line UFRJi007-A from a Brazilian familial amyotrophic lateral sclerosis patient.

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.

Generation of four patient-specific pluripotent induced stem cell lines from two Brazilian patients with amyotrophic lateral sclerosis and two healthy subjects.

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.

Vitamin D receptor gene FokI polymorphisms influence bone mass in adolescent football (soccer) players.

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.

Bone mass and breast milk calcium concentration are associated with vitamin D receptor gene polymorphisms in adolescent mothers.

Lactation-associated bone loss has been reported in adolescent mothers. Polymorphisms in the vitamin D receptor (VDR) gene may contribute to differences in the physiologic skeletal response to lactation in these mothers. We evaluated the influence of VDR gene polymorphisms ApaI, BsmI, and TaqI on bone mass, bone and calcium-related hormones, and breast milk calcium of lactating adolescents with habitually low calcium intake. Total body bone mineral content (TBMC), total body bone mineral density (TBMD), lumbar spine BMD (LSBMD), serum hormones [intact parathyroid hormone (iPTH), 25-hydroxyvitamin D, insulin-like growth factor-I (IGF1), prolactin, and estradiol), and breast milk calcium were measured in 40 lactating Brazilian adolescents (15-18 y), and compared by VDR genotype subgroups after adjustment for calcium intake and postmenarcheal and lactational periods. TBMD and LSBMD Z scores were -0.55 +/- 1.01 and -1.15 +/- 1.48, respectively. LSBMD was higher (21%; P < 0.05) in adolescents with the aa genotype (n = 5) compared with those with the AA genotype (n = 7). TBMC and IGF1 were higher (23 and 50%, respectively; P < 0.05) in adolescents with tt (n = 4) than those with TT (n = 29) and Tt (n = 7) genotypes. Breast milk calcium and serum iPTH were higher (24 and 80%, respectively; P < 0.05) in adolescents with bb (n = 8) compared with those with BB (n = 21) genotype. These results indicate that bone mass and breast milk calcium are significantly associated with VDR genotypes in lactating Brazilian adolescents. Those with aa and tt genotypes had a better bone status and those with bb genotype had greater breast milk calcium.

Polymorphic markers suggest a gene flow of CFTR gene from Sub-Saharan/Arabian and Mediterranean to Brazilian Population.

The analysis of 2 diallelic loci (M470V and T854T) and a microsatellite IVS8(T)n of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has shown different haplotype distribution in Brazilian cystic fibrosis (CF) chromosomes carrying different CF mutations. The DeltaF508 mutation was in absolute linkage disequilibrium with 1-1 haplotype (M470V-T854T). Most of DeltaF508 chromosomes (84%) were found to carry the IVS8-9T. The most frequent haplotypes IVS8-7T and 2-1 (M470V-T854T) were found associated with Non-DeltaF508 mutations. Although there is a remarkable linkage disequilibrium between these markers with CFTR locus, the mutations R334W (7T-1-2 and 7T-2-1) and the 3120 + 1G --> A (7T-1-2 and 9T-1-2) are associated with two different haplotypes probably introduced in the Brazilian population by migration. These findings suggest that recombination events from the original haplotype and gene flow among different ethnic groups (sub-Saharan and Mediterranean) might have resulted in CF mutations associated with different haplotypes by independent introductions.

Molecular analysis of 23 exons of the CFTR gene in Brazilian patients leads to the finding of rare cystic fibrosis mutations.

To define mutations present in 23 exons and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 95 patients from Rio de Janeiro, Brazil, we carried out single-strand conformation polymorphism analysis and automated direct sequencing. Mutation detection was achieved in 45% of the alleles presented, and complete genotyping (two mutated alleles) was accomplished in 34.7% of the patients. Twenty patients (21.1%) were found to carry only one mutation, whereas mutated alleles could not be observed in 42 patients (44.2%). Eleven mutations were found, of which four were characterized as rare mutations: P205S (1.05%), Y1092X (0.53%), S549R (0.53%), and S4X (0.53%). The DF508 mutation in this population sample showed a frequency of 28.42%. The low number of individuals (10 of 95; 10.5%) with compound heterozygous (DF508/non-DF508) genotypes could indicate the presence of another severe mutation leading to the premature death of these individuals. In 4 of the aforementioned 10 individuals with compound heterozygous genotypes, the D-7-2-1-2 (XV2c-KM19-IVS6a-TUB9-M470-T854) haplotype was defined.

Haplotype distribution of and linkage disequilibrium between four polymorphic markers near the CFTR locus in Brazilian cystic fibrosis patients.

To contribute to a better understanding of the origin and distribution of CFTR mutations in the Brazilian population, we have investigated the linkage between four polymorphic markers (XV2c, KM19, GATT, and TUB9) within or near the CFTR locus. The distribution of alleles for each polymorphism for both parental and cystic fibrosis (CF) chromosomes from Rio de Janeiro CF families were ascertained using a maximum-likelihood method. This same method was applied to study the distribution of the haplotypes defined by these markers. There was no significant association between the XV2c and KM19 loci on the parental and CF chromosomes. On the other hand, a strong association between GATT and TUB9 loci was observed on both CF and parental chromosomes, and striking linkage disequilibrium between the GATT-TUB9 pair and deltaF508 was observed (chi2 = 26.48, p < 0.0001). Remarkable linkage disequilibrium between the GATT-TUB9 marker pair and non-deltaF508 was also found (chi2 = 17.05, p < 0.0001). Our finding of a linkage disequilibrium between GATT-TUB9 and the CFTR locus could suggest that gene flow between different ethnic groups, mainly sub-Saharan and Mediterranean populations, with Brazilian populations could have resulted in some CF mutations originating on chromosomes that carried the GATT-TUB9 marker haplotype 7-2 (OR = 1.34 < 2.83 < 6.00; p = 0.0066).

Rastreamento da fibrose cística usando-se a análise combinada do teste de IRT neonatal e o estudo molecular da mutaçäo deltaF508 / Cystic fibrosis screening by two-tiered newborn IRT assay and deltaF508 mutation molecular analysis

Um total de 117 cartöes de rastreamento neonatal foi selecionado anonimamente para a avaliaçäo de fibrose cística (FC) pela análise da mutaçäo deltaF508 usando-se a técnica da reaçäo em cadeia da polimerase (PCR), seguida de eletroforese em gel de poliacrilamida (Page) e pela quantificaçäo da imunotripsina reativa (IRT, Delfia). Uma concentraçäo de IRT menor que 140ng/ml foi encontrada em 116 recém-nascidos. Entre estes foi detectado um heterozigoto deltaF508 com uma concentraçäo de IRT de 4,44ng/ml. Um dos 117 recém-nascidos era homozigoto näo-deltaF508 e apresentava uma IRT anormal de 410,7ng/ml. A média da concentraçäo da IRT diferia significativamente conforme este recém-nascido com IRT alterada era incluído ou excluído da amostragem populacional (n = 117, média = 8,207 ± 38,101; n = 116, média = 4,737 ± 6,597, respectivamente). Outra amostra de oito recém-nascidos, previamente rastreados pelo teste de IRT e com níveis elevados do analito, foi testada para a mutaçäo deltaF508. Em cinco deles a mutaçäo deltaF508 foi encontrada em um ou em ambos os cromossomos, correspondendo a 62,25 por cento dos recém-nascidos. De acordo com os resultados obtidos com triagem neonatal pela análise combinada IRT/DNA, pode-se concluir que o método só será eficiente se: a) forem excluídos os fatores que determinam os falso-positivos ou falso-negativos; e b) a detecçäo de outras mutações forem incluídas na análise dos resultados duvidosos