BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

Intestinal inflammation and the microbiota: Beyond diversity.

The recent manuscript entitled "Relationship between clinical features and intestinal microbiota in Chinese patients with ulcerative colitis" reported a difference in the intestinal microbiota of patients with ulcerative colitis according to the severity of the colitis. The influence of the intestinal microbiota on the development and progress of gastrointestinal disorders is well established. Besides the diversity in the microbiome, the presence of virulence factors and toxins by commensal bacteria may affect an extensive variety of cellular processes, contributing to the induction of a proinflammatory environment.

Avaliação de resíduos de agrotóxicos em amostras de farinha de trigo por espectrometria de massas / Evaluation of pesticide residues in wheat flour by mass spectrometry

Os cereais são uma importante fonte alimentícia e econômica, dentre eles se destaca o trigo e sua farinha. Durante o cultivo, visando o controle de pragas, frequentemente são utilizados os agrotóxicos, porém seu uso abusivo pode acarretar danos à saúde humana e ao meio ambiente. O trabalho teve como objetivo avaliar a presença de resíduos de agrotóxicos em amostras de farinha de trigo comercializadas no estado de São Paulo, utilizando o método QuEChERS (Rápido, Fácil, Barato, Efetivo, Robusto e Seguro) modificado, seguido de análise por CG-EM/EM e CLUE-EMAR para identificação e quantificação. No total, 124 ingredientes ativos (i.a.) estavam dentro dos critérios de aceitação para linearidade, limites de detecção e quantificação, exatidão e precisão (intervalo de confiança de 95%; k = 2). Os resultados de 25 amostras indicaram a presença de bifentrina, fenitrotiona, clorpirifós, deltametrina, lambda-cialotrina e pirimifós-metílico em farinha de trigo comum e os quatro últimos i.a. também foram detectados em farinha de trigo orgânica. O pirimifósmetílico foi detectado em 92% das amostras. Os i.a. encontrados nas amostras estavam abaixo do Limite Máximo de Resíduos (LMR) estabelecidos pela Agência Nacional de Vigilância Sanitária (Anvisa) para farinha de trigo e as amostras foram consideradas próprias para o consumo (AU).

Cereals are an important economic factor and food source, which wheat and its flour stand out. Pesticides are often employed to control harmful organisms, but their abusive use can cause damage to human health and the environment. This study aimed to evaluate the presence of pesticide residues in wheat flour samples commercialized in the state of São Paulo ­ Brazil, using the modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, followed by GC-MS/MS and UHPLC-HRMS analysis for identification and quantification. Thus, 124 active ingredients (a.i.) were within acceptable criteria for linearity, limits of detection and quantification, accuracy (trueness), and precision (95% confidence level; k = 2). The results for 25 wheat flour samples showed the presence of bifenthrin, fenitrothion, chlorpyrifos, deltamethrin, lambda-cyhalothrin, and pirimiphos-methyl, and the last four a.i. were also detected in organic wheat flour. Pirimiphos-methyl was detected in 92% of all samples. All compounds were found below the Maximum Residue Levels (MRL) established by Brazilian Health Regulatory Agency (Anvisa) for wheat flour and the samples were considered suitable for consumption (AU).

An Apparently Balanced Complex Chromosome Rearrangement Involving Seven Breaks and Four Chromosomes in a Healthy Female and Segregation/Recombination in Her Affected Son.

Duplication of the distal 1q and 4p segments are both characterized by the presence of intellectual disability/neurodevelopmental delay and dysmorphisms. Here, we describe a male with a complex chromosome rearrangement (CCR) presenting with overlapping clinical findings between these 2 syndromes. In order to better characterize this CCR, classical karyotyping, FISH, and chromosomal microarray analysis were performed on material from the patient and his parents, which revealed an unbalanced karyotype with duplications at 1q41q43 and 4p15.2p14 in the proband. The rearrangements, which were derived from a maternal balanced karyotype, included an insertion of a segment from the long to the short arm of chromosome 1, a balanced translocation involving chromosomes 14 and 18, and an insertion of a segment from the short arm of chromosome 4 into the derived chromosome 14. This study aimed to better define the clinical history and prognosis of a patient with this rare category of chromosomal aberration. Our results suggest that the frequency of CCR in the general population may be underestimated; when balanced, they may not have a phenotypic effect. Moreover, they emphasize the need for cytogenetic techniques complementary to chromosomal microarray for proper genetic counseling.

A novel variant in the COX15 gene causing a fatal infantile cardioencephalomyopathy: A case report with clinical and molecular review.

The cytochrome c-oxidase (COX) enzyme, also known as mitochondrial complex IV (MT-C4D), is a transmembrane protein complex found in mitochondria. COX deficiency is one of the most frequent causes of electron transport chain defects in humans. Therefore, high energy demand organs and tissues are affected in patients with mutations in the COX15 gene, with variable phenotypic expressiveness. We describe the case of a male newborn with hypertrophic cardiomyopathy and serum and cerebrospinal fluid hyperlacticaemia, whose exome sequencing revealed two variants in a compound heterozygous state: c.232G > A; p.(Gly78Arg), classified as likely pathogenic, and c.452C > G; p.(Ser151Ter), as pathogenic; the former never previously described in the literature.

Prevalence of Enteropathogens and Virulence Traits in Brazilian Children With and Without Diarrhea.

The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control (P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae-positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC.

Leigh syndrome in a patient with a novel C12orf65 pathogenic variant: case report and literature review.

Leigh syndrome is an early onset progressive disorder caused by defects in mitochondrial oxidative phosphorylation. Pathogenic variants in nuclear and mitochondrial genes are associated with the syndrome. Homozygous pathogenic variants in the C12orf65 gene impair the mitochondrial oxidative phosphorylation system. We describe a new case of Leigh syndrome caused by a novel pathogenic variant of the C12orf65 gene resulting in the lack of the Gly-Gly-Gln (GGQ) domain in the predicted protein, and review clinical and molecular data from previously reported patients. Our study supports that the phenotype caused by C12orf65 gene variants is heterogeneous and varies from spastic paraparesis to Leigh syndrome. Loss-of-function variants are more likely to cause the disease, and variants affecting the GGQ domain tend to be associated with more severe phenotypes, reinforcing a possible genotype-phenotype correlation.

Congenital afibrinogenemia in a patient with vascular abnormalities and a novel variant: clinical-molecular description and literature review.

: The objective is to report a patient with congenital afibrinogenemia and vascular abnormalities and also review the clinical and molecular issues. The female proband, diagnosed with congenital afibrinogenemia, was admitted at a hospital due to a hemorrhagic shock. Angiotomography revealed ectasias from ascending branch to the abdominal aorta, with multiple calcifications and atheroma. Clinical exome identified a homozygous novel pathogenic variant in FGG gene. In our review the main symptom, at diagnosis, was umbilical cord bleeding and the degree of clinical involvement varied from asymptomatic to severe. The FGA gene was the most affected and possible hot spots were observed. Variants considered as loss of function were the most frequent. The association of vascular abnormalities in a patient with congenital afibrinogenemia alerts for a closer follow-up of vascular issues in these patients.

Prevalence of enteropathogens and virulence traits in Brazilian children with and without diarrhea

The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control (P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae-positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC.

Qualitative, quantitative and genotypic evaluation of Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum isolated from individuals with different periodontal clinical conditions.

Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are strongly associated with periodontitis, and their evaluations are relevant to understand their role in the etiology and progression of periodontal diseases. In this study, the qualitative and quantitative detection of A. actinomycetemcomitans and F. nucleatum, as well as their genetic diversity, were evaluated in individuals with gingivitis, chronic periodontitis and periodontally healthy. In addition, the biotyping, serotyping, and prevalence of the ltx and cdt genes in A. actinomycetemcomitans were also determined. Subgingival biofilms obtained from gingivitis (70), periodontitis (75) and healthy (95) individuals were analyzed by cultures and PCR. Bacterial typing and presence of ltx and cdt genes in A. actinomycetemcomitans were also verified. DNA from A. actinomycetemcomitans and F. nucleatum was detected respectively, in 65.7% and 57.1% of gingivitis, 80% and 68% of periodontitis, and 57.8% and 37.8% of healthy. A. actinomycetemcomitans from gingivitis were biotypes I, II, IV, V, and X, and serotypes a, c, and e. In periodontitis, biotypes II, VI, and X, and serotypes a, b, and c were found. In healthy subjects, biotypes II and X, and serotypes b and c were found. The LTX and ltxA were observed in strains from gingivitis and periodontitis pockets. Subsequently, our data also showed no direct relationship between ltxA gene expression and leukotoxin gene 530-bp presence. On the other hand, cdt gene predominated during the inflammatory disease process. Our results strongly support a role of A. actinomycetemcomitans and F. nucleatum in advanced stage of periodontal disease.

A Novel MGP Gene Mutation Causing Keutel Syndrome in a Brazilian Patient.

Keutel syndrome is caused by mutations in the matrix gamma-carboxyglutamic acid (MGP) gene (OMIM 154870) and is inherited in an autosomal recessive fashion. It is characterized by brachydactyly, pulmonary artery stenosis, a distinctive facial phenotype, and cartilage calcification. To date, only 36 cases have been reported worldwide. We describe clinical and molecular findings of the first Brazilian patient with Keutel syndrome. Keutel syndrome was suspected based on clinical and morphological evaluation, so we sequenced the MGP gene using the TruSight One Sequencing Panel (Illumina). The obtained MGP gene sequence was then validated by Sanger sequencing. We identified a novel pathogenic homozygous variant of the MGP gene (c.2T>C; p.Met1Thr) confirming Keutel syndrome. Proper diagnosis of this syndrome is important for clinical management and is an indication for genetic counseling. Keutel syndrome should be suspected in patients with cartilage calcifications and brachydactyly when associated with a distinctive facial phenotype and pulmonary artery stenosis.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells

ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells.

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Pathogenicity and genetic profile of oral Porphyromonas species from canine periodontitis.

OBJECTIVE: In this study, the presence of the prtC and fimA genes involved in the pathogenicity of oral Porphyromonas spp. isolated from dogs with periodontitis and healthy, as well as their genetic diversity was investigated. DESIGN: Thirty-two Beagle dogs, 24 with periodontitis and 8 healthy were evaluated. Subgingival samples from only one gingival site of both groups were collected. Bacteria grown in anaerobiosis were identified by RAPID ID 32A kits. From each strain the respective DNA was obtained and used to genotyping by conventional PCR and AP-PCR. RESULTS: Dogs with periodontitis harbored 28 P. gulae, 2 P. creviocaricanis, 1 P. cangingivalis and 7 P. macacae; and from healthy dogs, 11 P. gulae and 5 P. circumdentaria. In P. gulae isolated from periodontal dogs the gene prtC was observed in 19 (67.85%) and in 7 (63.63%) from healthy dogs. P. gulae strains from periodontal dogs harbored either the gene fimA I or fimA II; while strains from healthy dogs harbored the gene fimA I, fimA II, fimA III or fimA IV, as well as 1 P. circumdentaria the gene fimA II. By AP-PCR strains were grouped in different clusters suggesting heterogeneity of these microorganisms. CONCLUSIONS: The results presented herein inform that Porphyromonas spp. isolated from dogs with and without periodontitis harbored the prtC and fimA genes and it could be a role in the establishment of the infectious process.

Organochlorine pesticides levels and associated factors in a group of blood donors in São Paulo, Brazil.

The use of organochlorine pesticides (OCPs) is a worldwide public health concern given that high levels of these compounds in humans and animals can lead to serious health problems. This study aimed to evaluate the levels of 15 organochlorine pesticides in the serum of 547 blood donors in the São Paulo metropolitan region (SPMR) in 2009 and to investigate factors associated with higher levels of these compounds. The OCPs were determined by gas chromatography with micro electron capture detection. Multiple ordinal logistic regression models were employed to determine the factors associated with higher levels. Only ß-HCH and p,p'-DDE had a significant number of samples above the limit of quantification. Factors associated with higher levels of ß-HCH were age within 26 to 35 years (OR = 3.1 [1.05-9.09]), age within 36 to 45 years (OR = 18.27 [6.89-48.91]), and female gender (OR = 0.44 [0.24-0.82] for men). p,p'-DDE levels were associated with age within 26 to 35 years (OR = 2.65 [1.20-3.45]), age within 36 to 45 years (OR = 4.59 [2.64-7.92]), female gender (OR = 0.86 [0.43-0.94] for men), and previous work with pesticides (OR = 2.88 [1.22-6.84]). Lower levels of p,p'-DDE were associated with an income of 3 to 5 minimum wages (OR = 0.38 [0.19-0.75]) and with intake of foods from animal origin up to twice a week (OR = 0.15 [0.03-0.72]) and more than twice a week (OR = 0.10 [0.02-0.47]). The results of this study indicate that the study group was not exposed to high levels of OCPs.

Alterations of Intestinal Microbiome by Antibiotic Therapy in Hospitalized Children.

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and a- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

High occurrence of Fusobacterium nucleatum and Clostridium difficile in the intestinal microbiota of colorectal carcinoma patients.

Colorectal carcinoma is considered the fourth leading cause of cancer deaths worldwide. Several microorganisms have been associated with carcinogenesis, including Enterococcus spp., Helicobacter pylori, enterotoxigenic Bacteroides fragilis, pathogenic E. coli strains and oral Fusobacterium. Here we qualitatively and quantitatively evaluated the presence of oral and intestinal microorganisms in the fecal microbiota of colorectal cancer patients and healthy controls. Seventeen patients (between 49 and 70 years-old) visiting the Cancer Institute of the Sao Paulo State were selected, 7 of whom were diagnosed with colorectal carcinoma. Bacterial detection was performed by qRT-PCR. Although all of the tested bacteria were detected in the majority of the fecal samples, quantitative differences between the Cancer Group and healthy controls were detected only for F. nucleatum and C. difficile. The three tested oral microorganisms were frequently observed, suggesting a need for furthers studies into a potential role for these bacteria during colorectal carcinoma pathogenesis. Despite the small number of patients included in this study, we were able to detect significantly more F. nucleatum and C. difficile in the Cancer Group patients compared to healthy controls, suggesting a possible role of these bacteria in colon carcinogenesis. This finding should be considered when screening for colorectal cancer.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children.

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.