Genetic analysis of osteogenesis imperfecta in a large Brazilian cohort.

INTRODUCTION: Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder caused by disruption of type I collagen synthesis. Previous Brazilian molecular OI studies have been restricted to case reports or small cohorts. The Brazilian OI Network (BOIN) is a multicenter study collecting clinical OI treatment data from five reference centers in three regions of Brazil. OBJECTIVE: To describe the molecular analysis of a large cohort of OI registered at BOIN. METHODS: Targeted next-generation sequencing (NGS) was performed at a centralized laboratory with the Ion Torrent platform, covering 99.6 % of the coding regions of 18 OI-associated genes. Clinical information was obtained from a clinical database. RESULTS: We included 156 subjects in the molecular analyses. Variants were detected in 121 subjects: 65 (53.7 %) in COL1A1, 42 (34.7 %) in COL1A2, 2 (1.7 %) in IFITM5, one (0.8 %) in CRTAP, three (2.5 %) in P3H1, two (1.7 %) in PPIB, four (3.3 %) FKBP10, one (0.8 %) in SERPINH1, and one (0.8 %) in TMEM38B. Ninety-one distinct variants were identified, of which 26 were novel. Of the 107 variants identified in COL1A1 and COL1A2, 24.5 % cause mild OI, while the remaining 75.5 % cause moderate, severe, or lethal OI, of which 49.3 % are glycine to serine substitutions. A single variant in FKBP10 (c.179A>C; p.Gln60Pro) was found in three unrelated and non-consanguineous participants living in the same geographic area in Northeast Brazil, suggesting a possible founder effect. CONCLUSION: Consistent with the literature, 88.4 % of the subjects had a variant in the COL1A1 and COL1A2 genes, with 10 % inherited in an autosomal recessive manner. Notably, one variant in FKBP10 with a potential founder effect requires further investigation. Data from this large cohort improves our understanding of genotype-phenotype correlations for OI in Brazil.

PADC nuclear track detector for ion spectroscopy in laser-plasma acceleration.

The transparent polymer polyallyl-diglycol-carbonate (PADC), also known as CR-39, is widely used as detector for heavy charged particles at low fluence. It allows for detection of single protons and ions via formation of microscopic tracks after etching in NaOH or KOH solutions. PADC combines a high sensitivity and high specificity with inertness towards electromagnetic noise. Present fields of application include laser-ion acceleration, inertial confinement fusion, radiobiological studies with cell cultures, and dosimetry of nuclear fragments in particle therapy. These require precise knowledge of the energy-dependent response of PADC to different ion species. We present calibration data for a new type of detector material, Radosys RS39, to protons (0.2-3 MeV) and carbon ions (0.6-12 MeV). RS39 is less sensitive to protons than other types of PADC. Its response to carbon ions, however, is similar to other materials. Our data indicate that RS39 allows for measuring carbon ion energies up to 10 MeV only from the track diameters. In addition, it can be used for discrimination between protons and carbon ions in a single etching process.

Measurements of indoor radon concentrations in the Santiago de Compostela area.

Galician soils are among those with the highest 222Rn exhalation rates in Spain. A year-round study of the indoor 222Rn concentration in buildings in the Santiago de Compostela area (Galicia, Northwest of Spain) was performed. The study is based on systematic samplings with active charcoal canisters, following a modified EPA 520/5-87-005 protocol. These measurements were complemented by others obtained using etched track dosimeters. Each data set follows a log-normal distribution, with a geometric mean of (253+/-3) Bq m(-3) for charcoal canisters and (285+/-2.5) Bq m(-3) for etched track detectors. After correcting for the different measuring conditions, the mean value of both methods differed by only 2%. A careful analysis of the seasonal dependence of our measurements did not reveal any significant seasonal variations in the 222Rn concentration. Parallel to these measurements, different meteorological parameters were recorded, which revealed a direct correlation between the indoor radon concentration and the outdoor temperature derivative with respect to time.

A Multicentric Brazilian Investigative Study of Copy Number Variations in Patients with Congenital Anomalies and Intellectual Disability.

Genomic imbalances are the most common cause of congenital anomalies (CA) and intellectual disability (ID). The aims of this study were to identify copy number variations (CNVs) in 416 patients with CA and ID from 5 different genetics centers within 4 different states by using the Multiplex Ligation-dependent Probe Amplification (MLPA) technique and to apply the chromosomal microarray (CMA) methodology in selected cases. The samples were analyzed by MLPA kits P064, P036, P070 and P250. Positive results were found in 97/416 (23.3%) patients. CMA was applied in 14 selected cases. In 6/14 (42.85%) patients, CMA detected other copy number variations not detected by the MLPA studies. Although CMA is indispensable for genotype refinement, the technique is still unfeasible in some countries as a routine analysis due to economic and technical limitations. In these cases, clinical evaluation followed by karyotyping and MLPA analysis is a helpful and affordable solution for diagnostic purposes.

Bax expression and apoptotic cell death in Fanconi anaemia peripheral blood lymphocytes.

OBJECTIVE: Deregulated apoptosis might be involved in some of the features of Fanconi anaemia (FA). The possibility that the pro-apoptotic Bax protein could be involved in an increased susceptibility to apoptosis in FA patients was investigated. MATERIALS AND METHODS: Intracellular Bax expression, Bcl-2 expression (an anti-apoptotic protein) and cell death were analysed in 26 FA peripheral blood lymphocyte samples. RESULTS: Most FA samples (69%) displayed increased levels of Bax and were more susceptible to both spontaneous apoptosis and mitogen activation-induced cell death. Two subgroups were identified: one presented elevated levels of Bax (n = 18), whereas the other (n = 8), had Bax levels lower than controls. Two subgroups based on Bcl-2 expression were also identified: one with normal and another with high Bcl-2 expression. No inverse correlation was found between Bcl-2 levels and Bax expression. A clear difference in susceptibility to induced cell death could be observed between control and FA samples. The best correlation was observed between high levels of Bax and mitogen-induced apoptosis of cells; these displayed characteristics of necrosis secondary to apoptosis, suggesting that the intrinsic apoptotic pathway was being activated. CONCLUSION: Despite increased susceptibility to cell death induction, there was no correlation between Bax levels, chromosome breakage, haematological parameters or androgen therapy. The importance of apoptosis and Bax expression in the clinical development of FA awaits clarification.

DNA extraction from fixed cytogenetic cell suspensions.

We developed a procedure for DNA extraction from small volumes of fixed cell suspensions previously prepared for conventional cytogenetic analysis. Good quality DNA was isolated with a fast and simple protocol using DNAzol reagent. This provided suitable DNA for various types of molecular analyses, including polymerase chain reaction, restriction fragment length polymorphism, denaturing high-performance liquid chromatography, and direct sequencing. This technique provides sufficient material for such test, which are important for diagnosis of neoplastic diseases in pediatric patients.

CODAS syndrome: a new distinct MCA/MR syndrome with radiological changes of spondyloepiphyseal dysplasia. Another case report.

We report on a young girl with psychomotor delay, cataracts, abnormally shaped teeth, malformed ears, and radiological findings of spondylo-epiphyseal dysplasia. The clinical picture resembles the CODAS syndrome described by Shebib et al. [Am J Med Genet 40: 88-93, 1991].

Molecular analysis of HPRT1(+) somatic cell hybrids derived from a carrier of an HPRT1 mutation responsible for Lesch-Nyhan syndrome.

Heterozygous carriers of HPRT1 mutations responsible for Lesch-Nyhan syndrome can be detected by analysis of somatic cell hybrids derived from peripheral blood lymphocytes and Hprt1-negative cells of rodent origin followed by selection in culture medium containing hypoxanthine, aminopterine, and thymidine (HAT). The parental origin of the X chromosome containing the normal HPRT1 allele in HPRT1(+) hybrid cell lines can be determined by molecular haplotyping attributable to highly polymorphic X-linked markers. We used this procedure to study a presumed carrier whose paternal active X chromosome always segregated in the cell hybrids derived from her. Conversely, her maternal X chromosome was systematically absent in most cell hybrids, or when present, it was inactive and coexisted with an active, paternal X chromosome. These results clearly demonstrated that the proband was a heterozygous carrier of a mutation responsible for HPRT1 deficiency.

Prenatal exposure to misoprostol and vascular disruption defects: a case-control study.

Prenatal exposure to misoprostol has been associated with Moebius and limb defects. Vascular disruption has been proposed as the mechanism for these teratogenic effects. The present study is a multicenter, case-control study that was designed to compare the frequency of prenatal misoprostol use between mothers of Brazilian children diagnosed with vascular disruption defects and matched control mothers of children diagnosed with other types of defects. A total of 93 cases and 279 controls were recruited in eight participating centers. Prenatal exposure was identified in 32 infants diagnosed with vascular disruption defects (34.4%) compared with only 12 (4.3%) in the control group (P<0.0000001). Our data suggest that prenatal exposure to misoprostol is associated to the occurrence of vascular disruption defects in the newborns.

Phaeohyphomycosis in El Salvador caused by Exophiala spinifera.

We identified Exophiala spinifera as the causal agent in a case of subcutaneous phaeohyphomycosis in El Salvador. Identification was based on the morphology of the fungus in tissue and the microscopic features of the culture obtained from the biopsy material. This case is the first of this type to be documented from Central America.

FRAXE mutation in mentally retarded patients using the OxE18 probe.

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. Although FRAXA and FRAXE are indistinguishable by means of conventional cytogenetics, they can now be delineated at the molecular level and provides the basis for a proper diagnosis. The screening for CGG amplifications in the FMR1 gene was based on standard protocols using EcoRI digests on Southern blots and hybridization with the StB12.3 probe. The FRAXE mutation was analyzed by digestion with HindIII and the filters were probed with OxE20. We present the results of 144 patients referred for fragile X testing but negative for the FMR1 gene trinucleotide expansion, that were also screened for the FMR2 expansion. For FRAXE mutation a molecular protocol for OxE18 probe was used, in the DNA samples digested with EcoRI on the same blots as those used for detection of FRAXA. None of the patients tested were positive for the FRAXE expansion. This technique was successfully established into our laboratory routine showing the practical use of testing for FRAXA and FRAXE in a large series of patients.

FRAXA screening in Brazilian institutionalized individuals with nonspecific severe mental retardation.

Individuals with mental disabilities are a heterogeneous group, mainly when we consider the etiology of mental retardation (MR). Recent advances in molecular genetics techniques have enabled us to unveil more about the molecular basis of several genetic syndromes associated with MR. In this study, we surveyed 85 institutionalized individuals with severe MR, 38 males and 47 females, by two molecular techniques, to detect CGG amplifications in the FMR1 gene. No FRAXA mutations were found in the FMR1 gene, reinforcing the low prevalence of Fragile X syndrome among institutionalized individuals with severe MR. We considered the PCR protocol used adequate for screening males with mental retardation of unknown etiology. The use of the Southern blot is still necessary for the decisive diagnosis of the Fragile X syndrome. To exclude chromosomal abnormalities associated with MR as a possible cause of the phenotype in these individuals, G-banded chromosome analysis was performed in all patients and 7.3% of chromosomal aberrations were found. Our results are similar to those reported previously and point to the necessity of expanding the molecular investigation toward other causes of MR, such as subtle chromosomal rearrangements, as suggested recent by a combination of fluorescence in situ hybridization (FISH) and PCR studies.

Urethral sonography in the diagnosis of penile and bulbar urethral stenosis.

Ultrasound was used in 25 patients for the study of penile (21) and bulbar (4) strictures and for the follow-up of 8 of these patients on whom dural urethroplasty was carried out. There was a good correlation with urethrography. Ultrasound has the added advantages of enabling study of the periurethral tissues without testicular irradiation, and safety and economy of the exploration.

Percutaneous catheter drainage of breast abscesses.

Experiences with ultrasonically guided percutaneous catheter drainage of breast abscesses (BA) are presented. The 12 cases were: 10 non-lactating women, one lactating women, and one HIV-positive man. The percutaneous procedure employed was successful in all cases. The mean duration of drainage was 5 days post-catheter insertion. All percutaneous drainages and subsequent follow-up were performed in the outpatient clinic. No recurrence of BA was observed. Percutaneous drainage of BA is a simple, effective and economical technique. Its use is recommended as an alternative to surgical drainage.

An episode of dioxin contamination in feedingstuff: the choline chloride case.

In 2000, as part of a survey programme, the German authorities detected high levels of dioxins in a choline chloride (CC) premix used as animal food component. The contaminated additive consisted of different products of mineral and vegetable origin acting as a carrier. The CC was manufactured in Belgium. The final product was produced in and distributed from a plant in Spain. The German authorities informed all European Community members of the incident. The Spanish Ministry of Agriculture, Fisheries and Food and the Regional Authorities immediately conducted a survey in collaboration with the Spanish Council for Scientific Research (CSIC) to determine and isolate the source of the contamination. Analysis of a large number of samples of pure CC, pine sawdust, almond shell and other substances currently used in the preparation of the premix confirmed the presence of a significant amount of polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans in mixtures in which pine sawdust was present. An analysis of the congener profiles revealed similarities with those found in technical pentachlorophenol (PCP) formulations. The conclusion was that PCP-contaminated sawdust as carrier for CC was source of dioxin contamination in feedingstuff.

[Fragile X syndrome confirmed by molecular analysis: a case-control study with pre and post-puberal patients]. / Síndrome do X frágil: estudo caso-controle envolvendo pacientes pré e pós-puberais com diagnóstico confirmado por análise molecular.

The fragile X syndrome (FRAXA) is the most common cause of inherited mental retardation. However, it has been frequently underdiagnosed in pediatric population. The characterization of the most significant pre and post-puberal clinical features observed among patients that are positive for the FMR-1 mutation, is useful as a screening tool for ordering the DNA test. Therefore, a screening program for FRAXA has been conducted in a sample of 104 mentally retarded individuals (92 males and 12 females), comprehending familial history and physical examination in order to determine the clinical characteristics. The molecular test for the disease was performed in all individuals. Seventeen patients (14 males) were positive for the FMR-1 mutation. Familial mental retardation and poor eye contact were the most common clinical findings with statistical significance (p<0.05) in FRAXA pre and post-puberal patients. The post-puberal patients presented, as opposed to the control group, large ears, broad forehead and macroorchidism.

[Down Syndrome--a clinical, cytogenetic and epidemiologic analysis of 165 patients]. / Síndrome de Down--análise clínica, citogenética e epidemiológica de 165 casos.

A clinical follow up of 165 Down Syndrome (DS) patients in an outpatient clinic programme at the Centro de Genética Médica (IFF - FIOCRUZ) was undertaken retrospectively. Clinical and laboratorial investigations were performed, such as cytogenetics and hematological analysis, thyroid hormones survey, abdominal ultrasound and cervical column X Rays. The clinical diagnosis of Down Syndrome was mostly performed during the first year of life, and 70% of all patients were under 4 years of age, being predominantly males. Trisomy 21 derived from non disjunction was found in 85% of the patients. The most common congenital malformation was cardiopathy (37.5%) and among all the clinical complications, repeated pneumonia could be evidenced in 30% of the patients, mainly during the first year of life. Leukopenia was observed in 14% of the patients and abdominal ultrasound scans allowed the early detection of biliary stones in 4.3% of the patients examined, a significative finding in the paediatric population. A prospective clinical programme aiming to anticipate the detection of clinical complications on at risk DS populations will fulfill its objectives and may act as a reducing factor in the infantile mortality rate.

[The role of vascular radiology in the diagnosis of "suspicious" images of the upper urinary tract]. / Papel de la radiología vascular en el diagnóstico de las imágenes "sospechosas" del tracto urinario superior.

Accurate diagnosis of "suspect" lesions from the Upper Urinary Tract (U.U.T.) is of major significance for implementing an appropriate treatment. There are many diseases and situations which may condition the occurrence of a suspect image, i.e., of a repletion defect of the U.U.T., in the U.I.V., the vascular imprints being just one of them. This paper contributes two cases where the suspect lesion was due to this cause and vascular radiology was decisive to reach a diagnosis.

Impact of the geological substrate on the radiological content of Galician waters.

Galicia (NW of Spain) is home to a highly-fractured soil rich in (238)U minerals, being the widest radon-prone area of the Iberian Peninsula. High precipitation levels confer a rich variety and abundance of both surface and groundwaters, which are extensively used for human consumption. Nevertheless, there exists no comprehensive body of information about the impact of the high environmental radioactivity on the radiological content of Galician waters. Measurements of (222)Rn, gross alpha/beta, (226, 224)Ra and (3)H activity were undertaken over a significant range of traditional springs, waters for spas and bottling plant wells. A seasonal survey was also performed for five network water suppliers to the largest Galician cities. The main outcome of this study has been the determination of statistical correlations between the water's radiological content and different environmental factors. Water measured at bottling plants reveal radiological values exceeding the U.E. limits, however this is eliminated in the industrial bottling process before reaching the consumer. Neither significant values nor seasonal variations were obtained for network waters.

An approach to the subslab depressurization remedial action in a high (222)Rn concentration dwelling.

Galicia (NW Spain) is a radon-prone area in the Iberian Peninsula. Measurements were carried out at a rural dwelling, with an annual average of radon concentration over 4000 Bq m(-3) and a maximum of 9000 Bq m(-3), found during a radon screening campaign held in the Autonomous Community of Galicia. We performed a detailed study to identify the main contamination source and the behaviour of the radon concentration, in which a linear dependence with temperature was verified, once corrected for relative humidity. We used different passive methods (charcoal canisters and two types of etched track detectors) as well as a radon concentration monitor that provided continuous measurement. Subsequent to this characterization, and in order to reduce the high radon concentration, a remedial action was developed using different passive and forced ventilation methods. A modified subslab depressurization technique was found to be the most effective remedy, providing a radon concentration reduction of around 96%. This method also has the advantages of being inexpensive and reliable over time.