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Objectives Recently, defects in the protein kinase mTOR (mammalian target of rapamycin) and its associated pathway have been correlated with hemimegalencephaly (HME). mTOR acts as a central regulator of important physiological cellular functions such as growth and proliferation, metabolism, autophagy, death, and survival. This study was aimed at identifying specific variants in mTOR signaling pathway genes in patients diagnosed with HME. Methods Using amplicon and whole exome sequencing (WES) of resected brain and paired blood samples from five HME patients, we were able to identify pathogenic mosaic variants in the mTOR pathway genes MTOR, PIK3CA, and DEPDC5. Results These results strengthen the hypothesis that somatic variants in PI3K‐Akt‐mTOR pathway genes contribute to HME. We also describe one patient presenting with a pathogenic variant on DEPDC5 gene, which reinforces the role of DEPDC5 on cortical structural changes due to mTORC1 hyperactivation. These findings also provide insights into when in brain development these variants occurred. An early developmental variant is expected to affect a larger number of cells and to result in a larger malformation, whereas the same variant occurring later in development would cause a minor malformation. Significance In the future, numerous somatic variants in known or new genes will undoubtedly be revealed in resected brain samples, making it possible to draw correlations between genotypes and phenotypes and allow for a genetic clinical diagnosis that may help to predict a given patient's outcome.

BackgroundThe Hereditary Breast and Ovarian Cancer Syndrome (HBOC) occurs in families with a history of breast/ovarian cancer, presenting an autosomal dominant inheritance pattern.BRCA1andBRCA2are high penetrance genes associated with an increased risk of up to 20-fold for breast and ovarian cancer. However, only 20–30% of HBOC cases present pathogenic variants in those genes, and other DNA repair genes have emerged as increasing the risk for HBOC. In Brazil, variants inATM, ATR, CHEK2, MLH1, MSH2, MSH6, POLQ, PTEN,andTP53genes have been reported in up to 7.35% of the studied cases. Here we screened and characterized variants in 21 DNA repair genes in HBOC patients.MethodsWe systematically analyzed 708 amplicons encompassing the coding and flanking regions of 21 genes related to DNA repair pathways (ABRAXAS1,ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51, TP53andUIMC1). A total of 95 individuals with HBOC syndrome clinical suspicion in Southeast Brazil were sequenced, and 25 samples were evaluated for insertions/deletions inBRCA1/BRCA2genes. Identified variants were assessed in terms of population allele frequency and their functional effects were predicted through in silico algorithms.ResultsWe identified 80 variants in 19 genes. About 23.4% of the patients presented pathogenic variants inBRCA1, BRCA2andTP53, a frequency higher than that identified among previous studies in Brazil. We identified a novel variant inATR, which was predicted as pathogenic by in silico tools. The association analysis revealed 13 missense variants inABRAXAS1, BARD1, BRCA2, CHEK2, CDH1, MLH1, PALB2,andPMS2genes, as significantly associated with increased risk to HBOC, and the patients carrying those variants did not present large insertions or deletions inBRCA1/BRCA2genes.ConclusionsThis study embodies the third report of a multi-gene analysis in the Brazilian population, and addresses the first report of many germline variants associated with HBOC in Brazil. Although further functional analyses are necessary to better characterize the contribution of those variants to the phenotype, these findings would improve the risk estimation and clinical follow-up of patients with HBOC clinical suspicion.

The effluent discharged by oil refineries is known for its toxicity caused by its inorganic and organic chemicals. The biological effects of the effluent from a treated oil refinery on the bivalve filter feeder of Corbicula fluminea were assessed. DNA damages were assessed through comet assay, the biochemical parameters were measured based on the activity of enzymes such as catalase (CAT) and glutathione S-transferase (GST). Animals were exposed to treatments with effluent for 6, 24 and 96 h. Test animals were allowed to recover from the exposure period for 24, 72 and 144 h. Although animals exposed to the effluent for 96 h presented genotoxicity, they were fully recovered 144 h after the exposure period was over. The CAT activity of animals exposed to the effluent significantly reduced after 24 h of exposure. However, GST activity increased after 96 h of exposure, and its rate remained high after 144 h of recovery; therefore, there was protective GST activity. The damage detected after 96 h of exposure could have been caused by the polycyclic aromatic hydrocarbons (PAH) herein described as DNA intercalating agents. Other compounds such as monoaromatic ethylbenzene, toluene and xylenes (BTEX) were also detected in the assessed effluent. Our study is the first to use the comet assay as genotoxicity cytogenetic biomarker to evaluate the overall recovery ability of bivalve mollusks, mainly of C. fluminea, after their exposure to effluent discharged by oil refineries. Further studies can help better understanding the responses from such exposure and improve knowledge about the persistence of certain disturbances even after contamination is over.

Due to the utilization of milk proteins such as whey protein (WP) and casein as sports nutrition ergogenic aids, the present study investigated the effects of the association of WP and casein in a ratio of 80:20, a similar ratio of human breast milk, on blood branched-chain amino acid (BCAA) profiles, markers of protein metabolism and delayed onset muscle soreness (DOMS), after a single bout of resistance exercise. A double-blind, crossover and acute study was carried out with ten men (age 29 ± 8 years, BMI: 25.4 ± 2.9 kg/m2, 77 ± 12 kg, 1.74 ± 0.09 m), each one consumed the following supplements randomly, one per session: WP, CAS (casein), WP/CAS (80% WP/20% CAS), CAS/WP (80% CAS/20% WP) and PLA (placebo). They were also subjected to the following evaluations: the one repetition maximum (1RM) test, resistance training session, blood extraction during each session to determine the BCAA profile, two food records, 3-day evaluation of DOMS (24 h, 48 h and 72 h) and nitrogen balance in each treatment. The intervention resulted in similar nitrogen urinary, creatinine and urea plasma levels and showed a positive nitrogen balance in all the trials. Regarding the BCAAs, the peak occurred at 60 min post-ingestion and remained higher until 120 min for WP, WP/CAS and CAS/WP. The DOMS was significantly lower for WP, WP/CAS and CAS/WP compared to the CAS and PLA treatments. There were no advantages in the association of WP and CAS in the BCAAs profile when compared to WP itself, but it induced a lower DOMS compared to CAS and PLA (Clinical Trial registration number: clinicaltrials.gov, NCT04648384).

A Síndrome Hereditária do Câncer de Mama e/ou Ovário (HBOC) é caracterizada por famílias com história de câncer de mama e/ou ovário e associada a um padrão de herança autossômica dominante. Esta síndrome abrange cerca de 10% e 15% de todos os casos de câncer de mama e de ovário, respectivamente. Os genes BRCA1 e BRCA2 são genes de alta penetrância associados ao aumento de até 20x no risco para o desenvolvimento do câncer de mama e/ou ovário. Entretanto, apenas 20-30% dos casos de HBOC apresentam variantes patogênicas nestes genes e estudos têm evidenciado a contribuição de outros genes ligados às vias de reparo do DNA para o aumento de risco para HBOC. Na população brasileira, variantes em genes como ATM, ATR, CHEK2, MLH1, MSH2, MSH6, POLQ, PTEN e TP53, têm sido relatadas em até 7,35% dos casos estudados. Assim, o objetivo destes estudo foi realizar a pesquisa e caracterização de variantes em 21 genes relacionados à HBOC (ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, FAM175A, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51, TP53 e UIMC1) em 95 indivíduos diagnosticados com suspeita clínica de HBOC no Sudeste brasileiro. Foram identificadas 78 variantes patogênicas ou de significado incerto com conflito de informações sobre patogenicidade na literatura, em 19 dos 21 genes analisados. Cerca de 23,4% dos pacientes apresentaram variantes patogênicas nos genes BRCA1, BRCA2 e TP53, frequência maior que a identificada por outros estudos na população brasileira. No entanto, 83,3% das variantes identificadas são de significado incerto. A análise de associação identificou 13 destas VUS, em 8 genes, como significativamente associadas ao risco para HBOC. Os pacientes portadores destas variantes não apresentaram alterações no número de cópias para os genes BRCA1/BRCA2. Cinco variantes no BRCA1 foram identificadas como sendo segregadas em haplótipo, mas este não foi associado ao risco para HBOC. Duas variantes nos genes FAM175A e UIMC1 apresentaram alta frequência nos casos estudados e pacientes duplo heterozigotos apresentaram deficiência no reparo de danos causado após irradiação. Duas variantes no gene PALB2 associadas ao risco não afetaram a interação com BRCA1 e apresentaram atividade de reparo similar à proteína selvagem. No entanto, uma variante no CHEK2 levou à instabilidade da proteína e diminuição nos níveis de ativação de CHK2. Embora não associadas ao risco, duas variantes identificadas nos genes PMS2 e BRIP1 foram classificadas como possivelmente patogênicas. Esta variante no gene BRIP1 apresentou sensibilidade ao tratamento à mitomicina C, evidenciando uma deficiência na função desta proteína. Estudos como este evidenciam como outros genes envolvidos em vias de reparo do DNA podem estar contribuindo para a patogênese do HBOC. A caracterização destas variantes é imprescindível para estimar os riscos associados e assim possibilitar o acompanhamento clínico e aconselhamento genético adequados para pacientes diagnosticados clinicamente com HBOC, que, em sua maioria, permanecem sem um diagnóstico molecular elucidado. The Hereditary Breast and Ovarian Cancer Syndrome (HBOC) is characterized by families with a history of breast/ovarian cancer with an autosomal dominant inheritance patern. This syndrome covers about 10% to 15% of all cases of breast and ovarian cancer, respectively. The BRCA1 and BRCA2 genes are high penetrance genes associated with an increased risk of up to 20x for breast and ovarian cancer. However, only 20-30% of HBOC cases present pathogenic mutations in those genes, and other DNA repair genes have emerged as increasing the risk for HBOC. In the Brazilian population, mutations in genes such as ATM, ATR, CHEK2, MLH1, MSH2, MSH6, POLQ, PTEN and TP53 have been reported in up to 7.35% of the studied cases. The aim of this study was to investigate and characterize mutations in 21 HBOC-related genes (ATM, ATR, BARD1, BRCA1, BRCA1, BRIP1, CDH1, CHEK2, FAM175A, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51, TP53 and UIMC1) in 95 individuals clinically diagnosed with HBOC in the Southeastern Brazil. We identified 78 pathogenic mutations and variants of uncertain significance with conflicting data on pathogenicity for 19 of the 21 genes analysed. About 23.4% of the patients presented pathogenic mutations in BRCA1, BRCA2 and TP53, a frequency higher than that identified by other studies in the Brazilian population. However, 83.3% of the identified variants are of uncertain significance. Five BRCA1 variants were identified as being segregated in haplotype, but this was not associated with risk for HBOC. Two variants in FAM175A and UIMC1 genes presented high frequency in the studied cases and when in double heterozygosity, presented deficiency in the repair of DNA damage after irradiation. The association analysis showed 13 VUS in 8 genes, as significantly associated with increased risk for HBOC, and the patients that carry those VUS did not present copy number variations for BRCA1/BRCA2 genes. Two PALB2 variants associated with risk did not affect the interaction with BRCA1 and presented repair activity similar to the wild type protein. However, one variant in CHEK2 led to protein instability and decreased levels of CHK2 activation. Although not associated with an increased risk, two variants identified in PMS2 and BRIP1 were classified as possibly pathogenic. The BRIP1 variant showed sensitivity to mitomycin C treatment, evidencing a deficiency in the protein function. Data such as this show how much other DNA repair genes may be contributing to the HBOC pathogenesis, emphasizing how essential the characterization of variants of unknow significance is for estimating the risks and thus enabling adequate clinical follow-up and genetic counseling for patients clinically diagnosed with HBOC, which, for the most part, remain without an elucidated molecular diagnosis.

While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

Made available in DSpace on 2018-11-26T15:28:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundacao deAmparo e Pesquisa do Estado do Piaui (FAPEPI) The aim of this study was to evaluate the oxidative parameters of erythrocytes and genotoxicity in leukocytes of patients with breast cancer. Oxidative parameters were detected by spectrophotometry and genotoxic damage by single cell gel electrophoresis. Twenty-eight women with breast cancer were monitored before chemotherapy and after the second and fourth cycles of therapy with cyclophosphamide and doxorubicin. After the fourth cycle, increases (P < 0.05) in the reactive substances to thiobarbituric acid levels, nitrite content, and superoxide dismutase activity and high rates of DNA damage in leukocytes were observed when compared with healthy women group and baseline levels. Similarly, after the second cycle, the same parameters were increased (P < 0.05) when compared with baseline levels. Increase in catalase activity was detected only after the fourth cycle and reduced glutathione levels and glutathione peroxidase activity were decreased in all cycles when compared with healthy women, as well as after the second and fourth chemotherapy cycles compared to baseline (P < 0.05). Patients with breast cancer presented an indicative of oxidative stress before, during, and after chemotherapy, as well as increased genotoxic damage in all stages of treatment, demonstrating the clinical applicability of this investigation. Univ Fed Piaui, Lab Genet Tox, Postgrad Program Pharmaceut Sci, BR-64049550 Teresina, PI, Brazil State Univ Sao Paulo, Postgrad Program Genet, BR-14049900 Ribeirao Preto, SP, Brazil Univ Fed Piaui, Dept Physiol & Biophys, Postgrad Program Pharmaceut Sci, Lab Expt Cancerol, BR-64049550 Teresina, PI, Brazil Univ Alabama, Dept Chem, Huntsville, AL 35899 USA Univ Fed Piaui, Lab Res Expt Neurochem, Postgrad Program Pharmaceut Sci, BR-64049550 Teresina, PI, Brazil State Univ Sao Paulo, Postgrad Program Genet, BR-14049900 Ribeirao Preto, SP, Brazil

A audição representa a principal fonte para o aprendizado da fala e linguagem durante a infância e a surdez e a privação de estímulos auditivos pode implicar em dificuldades emocionais e sociais àqueles indivíduos afetados. Aproximadamente 360 milhões de pessoas sofrem de perda auditiva no mundo, o que corresponde a 5,3% da população mundial. A surdez pode se desenvolver em decorrência de causas genéticas (hereditárias), não-genéticas e ambientais. As infecções pré-natais e a exposição a ruídos constituem as causas ambientais mais comuns. Já a surdez hereditária, constitui o transtorno neurossensorial mais comum em humanos, com uma prevalência de 1:1000 nascidos vivos. Mais de 70% dos casos de surdez hereditária constituem casos não-sindrômicos, destes cerca de 70% cursam com surdez congênita ou pré-lingual. Na maioria dos casos, a perda auditiva hereditária é neurossensorial, heterogênea, com diferentes padrões de herança e com uma grande quantidade de genes envolvidos. Estudos têm demonstrado o importante papel dos genes GJB2, GJB6 e SLC26A4 na fisiologia do ouvido interno e alterações nestes genes têm sido relatadas como causa da surdez hereditária. Desta forma, o objetivo deste estudo foi investigar a base genética e o papel do gene SLC26A4 na perda auditiva neurossensorial (PANS) nãosindrômica pré-lingual em pacientes atendidos pelo serviço de Genética Médica do Hospital das Clínicas de Ribeirão Preto. Para isso, uma série de 88 casos foi investigada quanto a características clínicas e moleculares. A amostra abrangeu indivíduos de ambos os sexos, com idade de 2 a 63 anos, provenientes de 88 famílias diferentes, assistidos durante o período de 2003 a 2013. As amostras foram triadas pela técnica de High Resolution Melting (HRM) e em seguida levadas para o seqüenciamento para caracterização das alterações. Na série de casos estudada, 23,9% (21/88) dos pacientes portadores de surdez neurossensorial não-sindrômica pré-lingual evidenciaram alterações nos genes GJB2, GJB6 e SLC26A4 sugeridas como patogênicas. A prevalência de alterações no gene SLC26A4 foi de 28,4% (25/88), não relacionada à Síndrome do Aqueduto Vestibular Alargado (SAVA). Dentre as 11 alterações encontradas neste gene, três constituem mutações não descritas: p.Gly139Arg, p.Ile254Val, p.Asn382Lys. Os genótipos mais freqüentes neste estudo foram a c.35delG/c.35delG no gene GJB2 (5/88), a dupla heterozigose com o gene GJB6 c.35delG/del(GJB6-D13S1854) (3,4%) e chr7:g.107301238C>G/wt no gene SLC26A4 (10,2%). Entretanto, apenas 19,3% dos indivíduos apresentaram genótipos sugeridos como responsáveis pelo fenótipo estudado. Alterações particulares no gene SLC26A4 podem sugerir a explicação para a surdez genética para aproximadamente 9,1% destes casos. Destes, cinco casos de heterozigose preditas como patogênicas (p.Ile300Leu; p.Asn324Tyr e p.Asn382Lys), dois casos de heterozigose composta (chr7:g.107301201T>C/chr7:g.107301238C>G e chr7:g.107301238C>G/p.Gly139Arg) e um caso de dupla heterozigose com GJB2 (chr7:g.107301238C>G/c.35delG). Isto ressalta a importância do gene SLC26A4 para o diagnóstico molecular de surdez hereditária e reforça a sua potencial contribuição para o processo de aconselhamento genético. Entretanto, nossos dados sugerem a necessidade de testes funcionais a fim de elucidar o papel destas alterações para o estabelecimento do fenótipo, como também, a presença de outros genes ou regiões envolvidas naqueles casos em que mutações monoalélicas não foram suficientes para justificar o fenótipo. The hearing is the main source for learning speech and language during childhood and deafness and deprivation of auditory stimuli can result in emotional and social difficulties to those affected individuals. Approximately 360 million people suffer from hearing loss worldwide which corresponds to 5.3% of the world population. Deafness may develop due to genetic (hereditary), non-genetic and environmental causes. Prenatal infections and exposure to noise are the most common environmental causes. Hereditary deafness is the most common sensorineural disorder in humans, with a prevalence of 1:1000 live births. More than 70% of hereditary deafness cases are nonsyndromic, about 70% of these occur with congenital or prelingual deafness. In most cases, inherited sensorineural hearing loss is heterogeneous, with different patterns of inheritance and with a large number of genes involved. Studies have shown the important role of genes GJB2, GJB6 and SLC26A4 in the physiology of the inner ear and changes in these genes have been reported as cause of hereditary hearing loss. Thus, the aim of this study was to investigate the genetic basis and the role of SLC26A4 gene in non-syndromic prelingual sensorineural hearing loss (SNHL) in patients enrolled in the Medical Genetics Service of Hospital das Clínicas de Ribeirão Preto. For this, a series of 88 cases were submitted to a clinical and molecular investigation. The sample consisted of individuals of both sexes, aged 2-63 years from 88 different families, assisted during the period 2003-2013. The samples were screened by the technique of High Resolution Melting (HRM) and then taken for sequencing to characterize the mutations. In the series of cases studied, 23.9% (21/88) of patients with non-syndromic prelingual sensorineural deafness showed variants in genes GJB2, GJB6 and SLC26A4 suggested as pathogenic. The prevalence of mutations in the SLC26A4 gene was 28.4% (25/88), not related to non-syndromic EVA. Among the 11 mutations found in this gene, three are reported as novel mutations: p.Gly139Arg, p.Ile254Val, p.Asn382Lys. The most frequent genotypes found in this study were the c.35delG/c.35delG in GJB2 gene (5/88), the double heterozygosity with GJB6 gene c.35delG/del(GJB6-D13S1854) (3,4%) and chr7:g.107301238C>G/wt in the SLC26A4 gene (10,2%). However, only 19.3% of subjects presented genotypes suggested as responsible for the studied phenotype. Particular mutations in the SLC26A4 gene may suggest the explanation for the genetic deafness to approximately 9.1% of these cases. Of these, five cases of heterozygosity predicted as pathogenic (p.Ile300Leu; p.Asn324Tyr and p.Asn382Lys), two cases of compound heterozygosity (chr7:g.107301201T>C/chr7:g.107301238C>G and chr7:g.107301238C>G/p.Gly139Arg) and one case of double heterozygosity with GJB2 gene (chr7:g.107301238C>G/c.35delG). Those data highlights the importance of the SLC26A4 gene for molecular diagnosis of hereditary hearing loss and give strength to its potential contribution to the genetic counseling process. However, our data suggest the need for functional tests in order to elucidate the role of these changes to the phenotype, as well as the presence of other genes or regions involved in those cases that monoallelic mutations were not sufficient to justify the phenotype.

Mutagenic and antimutagenic activities of the medicinal plant Duguetia furfuracea were assessed using SMART/wing and ring-X-loss tests. For the ring-X-loss test, 2- to 3-day-old Drosophila melanogaster ring-X-lineage males and virgin ywsn³ females received D. furfuracea infusion at doses of 0.085, 0.042, or 0.014 g/mL for 24 h. We found that D. furfuracea did not produce any mutagenic effects in D. melanogaster germinative cells. The somatic cells of D. melanogaster were analyzed using the SMART/wing test involving three lineages - mwh, flr³, and ORR - and the same doses of D. furfuracea infusion employed in the ring-X-loss test, as well as 20 mM urethane. The results of both standard (ST) and high bioactivation (HB) crosses showed absence of mutagenic activity of D. furfuracea. In contrast, in both ST and HB crosses, we observed a modulatory effect of D. furfuracea against the genotoxic activity of urethane.