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.

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.

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.

(222)Rn concentration in public secondary schools in Galicia (Spain).

In the framework of a (222)Rn screening campaign that was carried out in 58 public secondary schools in Galicia (NW Spain), the largest radon-prone area in the Iberian Peninsula, a positive correlation between indoor (222)Rn concentration and outdoor gamma exposure rate was obtained. A new approach to the data acquisition in screening surveys was tested, improving the performances of this type of study and gathering useful data for future remedial actions. Using short-period detectors (charcoal canisters) firstly, in order to detect places showing (222)Rn concentrations over 400Bqm(-3), the number of locations to be measured with long-period detectors (etched track detectors) is reduced. In this screening campaign, 34% of the schools surveyed presented at least one site exceeding the 400Bqm(-3) recommended action level established by the EU, and 15% had at least one site with (222)Rn values over 800Bqm(-3). The maximum value recorded was 2084+/-63Bqm(-3). These results are discussed and compared with data obtained in schools of several countries with similar geology. Seven schools were also studied for seasonal variations of (222)Rn activity concentration. The results were not conclusive, and no significant correlation between season and (222)Rn concentration was established. Finally, a continuous (222)Rn concentration monitor was placed in the secondary school exhibiting a mean value of the (222)Rn concentration very close to 400Bqm(-3). Maximum (222)Rn concentration values were found to occur at times when the school was unoccupied.

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.

Uncertainty assessment of polychlorinated dibenzo-p-dioxins and dibenzofuran and dioxin-like polychlorinated biphenyl analysis in stationary source sample emissions in accordance with the impending European standard EN-1948 using fly ashes.

The analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) present in stack gas emissions and solid residues from incinerators will be mandatory in the foreseeable future. European standard EN-1948 is in the process of being updated through the addition of a new Part 4 related to the analysis of the 12 dl-PCBs. Therefore, either a comprehensive and reliable method capable of analyzing all of these 29 compounds (12 dl-PCBs and 17 2,3,7,8-PCDD/Fs) needs to be developed, or the existing PCDD/F analytical procedure must be adapted to include the dl-PCBs. This study has taken the latter approach of modifying PCDD/F methodology and in particular the fractionation step, by isolating dioxins and dl-PCBs into separate fractions ready for high resolution gas chromatography coupled to high resolution mass spectrometry (HRGC/HRMS) analysis. Results obtained from the analysis of Certified Reference Materials (CRM-490 and CRM-615) and fly ashes from the European Committee for Standardization (CEN) intercalibration study demonstrated that the proposed methodology is appropriate to determine the dl-PCBs in accordance with the impending European standard EN-1948. Uncertainty values obtained during the validation of the analytical methodology were 13% total I-TEQ (International Toxic Equivalent) for PCDD/Fs and 31% total WHO-TEQ (World Health Organization Toxic Equivalent) in the case of dl-PCBs. In addition, 'real' samples such as emissions and fly ashes were successfully analyzed following the proposed analytical method.

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.

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.

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.

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.

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.

Identification of novel mutations in SHH and ZIC2 in a South American (ECLAMC) population with holoprosencephaly.

Holoprosencephaly (HPE) is genetically heterogeneous with four genes, SIX3, SHH, TGIF, and ZIC2 that have been identified to date and that are altered in 12% of patients. To analyze this prevalence in a South American population-based sample (57 HPE cases in 244,511 live and still births or 1 in 4300), we performed a mutational study of these genes in 30 unrelated children (26 newborns and 4 non-newborns) with HPE being ascertained by ECLAMC (Latin American Collaborative Study of Congenital Malformations). We identified three novel mutations: two were missense mutations of the SHH gene (Cys183-->Phe; His140-->Pro); the third mutation was a 2-bp deletion in the zinc-finger region of the ZIC2 gene. These molecular results explained 8% (2/26 newborn samples) of the HPE cases in this South American population-based sample, a proportion similar to our previously published data from a collection of cases.

[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.

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.

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.

[Respiratory complications in hematopoietic stem cell transplantation. What does radiology contribute?]. / Complicaciones respiratorias en el trasplante de progenitores hematopoyéticos. Qué aporta la radiología?

OBJECTIVE: To determine the incidence of respiratory complications and their manifestations in both conventional radiology and high-resolution computerized tomography (HRCT) in a series of patients who had undergone blood progenitor cell transplantation (BPCT). The objective was to evaluate whether the radiological findings associated with post-transplantation time can be useful for establishing the differential diagnosis. PATIENTS AND METHODS: A study was undertaken of a total of 108 consecutive patients who had undergone BPCT and were alive one year after; from these patients a selection was made of those who had some respiratory complications during the first year after transplantation. Complications were classified in three different groups on the basis of time elapsed since transplantation (early, intermediate, and late stages). Chest X-ray films of each patient were examined and in 17 cases the study was completed with HRCT. These findings were correlated with both definitive diagnosis and time elapsed since BPCT. The following procedures were useful for diagnostic confirmation: blood culture, sputum culture, fibrobronchoscopy with bronchoalveolar lavage or lung biopsy, biopsy by other methods, necropsy, or clinical course after empirical therapy. RESULTS: Thirty-three out of the 108 patients undergoing BPCT had some form of respiratory complication during the first year after transplantation. The most common radiological pattern both in the chest X-ray and in HRCT was alveolar consolidation. Other findings included ground-glass appearance, interstitial pattern, pulmonary nodules, and pleural effusion. The diagnoses in relation to the different stages of transplantation were: a) early stage: three cases of heart failure, two cases of alveolar hemorrhage, two cases of pulmonary aspergillosis and three cases of undocumented complications; b) intermediate stage: four cases of cytomegalovirus pneumonia, one case of pulmonary aspergillosis, two cases of bacterial pneumonia, and two cases of undocumented pneumonitis; c) late stage: four cases of bacteriologically documented pneumonia, two cases of pneumonitis with an unidentified agent, two cases of graft-versus-host disease, one case of pulmonary aspergillosis and two complications without established diagnosis. CONCLUSIONS: The relationship between radiological findings and time elapsed since transplantation of blood precursor cells is very useful for establishing the diagnosis of pulmonary complications. High resolution computerized tomography is useful for detecting unnoticed lesions in conventional X-ray, and for diagnosing bronchiolitis obliterans and some fungal lesions.

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.

A de novo complex chromosomal rearrangement with nine breakpoints characterized by FISH in a boy with mild mental retardation, developmental delay, short stature and microcephaly.

A de novo complex chromosome rearrangement (CCR) involving chromosomes 1, 6, 7, 15 and Y was detected in a boy with mental retardation, short stature, and microcephaly. Fluorescence in situ hybridisation (FISH) with whole chromosome painting libraries, band-specific cosmids and telomeric probes was essential for the characterisation of the rearrangement. The CCR was shown to be the result of at least nine chromosomal breaks and involved the alternating insertion of two segments of the short arm of chromosome 1 and two segments of the long arm of chromosome 6 into a novel derived chromosome 7. A non-reciprocal translocation between the distal short arm of the same chromosome 7 and the distal long arm of the Y chromosome was also found, together with a paracentric inversion of the long arm of chromosome 15. The only detectable imbalance was a deletion of the heterochromatic Yq telomeric region. FISH investigations in this case have revealed an additional complexity in this CCR, which has implications for reproductive risk assessment and genetic counselling.

Cystic fibrosis: low frequency of DF508 mutation in 2 population samples from Rio de Janeiro, Brazil.

Blood samples from 44 unrelated cystic fibrosis (CF) patients from Rio de Janeiro, Brazil, were analyzed for the 8 European CF mutations. Six homozygous and 15 heterozygous carriers of the DF508 mutation were found, corresponding to 47.7% of CF patients (allele frequency 0.3068). The G542X and G551D mutations were also observed with allele frequencies of 0.0227 and 0.0114, respectively. An analysis of the DF508 mutation in 291 randomly chosen, healthy individuals was performed, and only 3 heterozygous carriers were identified. These results show that the frequency of the DF508 allele in Rio de Janeiro is much lower than the world average; this may be due to the extremely heterogeneous ethnic admixture of the study population. By combining the results of these 2 different samples (CF patients and random population) and admixture data from Rio de Janeiro, we can estimate the CF incidence in this population to be 1:3542 individuals. However, taking into account the Rio de Janeiro ethnic admixture, we can find an estimate of 1:6902 individuals.