Systematic reanalysis of genomic data improves quality of variant interpretation.

As genomic sequencing expands, so does our knowledge of the link between genetic variation and disease. Deeper catalogs of variant frequencies improve identification of benign variants, while sequencing affected individuals reveals disease-associated variation. Accumulation of human genetic data thus makes reanalysis a means to maximize the benefits of clinical sequencing. We implemented pipelines to systematically reassess sequencing data from 494 individuals with developmental disability. Reanalysis yielded pathogenic or likely pathogenic (P/LP) variants that were not initially reported in 23 individuals, 6 described here, comprising a 16% increase in P/LP yield. We also downgraded 3 LP and 6 variants of uncertain significance (VUS) due to updated population frequency data. The likelihood of identifying a new P/LP variant increased over time, as ~22% of individuals who did not receive a P/LP variant at their original analysis subsequently did after 3 years. We show here that reanalysis and data sharing increase the diagnostic yield and accuracy of clinical sequencing.

mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis.

Novel personalized therapies for cystic fibrosis: treating the basic defect in all patients.

Cystic fibrosis (CF) is the most common genetic life-shortening condition in Caucasians. Despite being a multi-organ disease, CF is classically diagnosed by symptoms of acute/chronic respiratory disease, with persistent pulmonary infections and mucus plugging of the airways and failure to thrive. These multiple symptoms originate from dysfunction of the CF transmembrane conductance regulator (CFTR) protein, a channel that mediates anion transport across epithelia. Indeed, establishment of a definite CF diagnosis requires proof of CFTR dysfunction, commonly through the so-called sweat Cl(-) test. Many drug therapies, including mucolytics and antibiotics, aim to alleviate the symptoms of CF lung disease. However, new therapies to modulate defective CFTR, the basic defect underlying CF, have started to reach the clinic, and several others are in development or in clinical trials. The novelty of these therapies is that, besides targeting the basic defect underlying CF, they are mutation specific. Indeed, even this monogenic disease is influenced by a large number of different genes and biological pathways as well as by environmental factors that are difficult to assess. Accordingly, every person with CF is unique and so functional assessment of patients' tissues ex vivo is key for diagnosing and predicting the severity of this disease. Of note, such assessment will also be crucial to assess drug responses, in order to effectively treat all CF patients. It is not because it is a monogenic disorder that personalized treatment for CF is much easier than for complex disorders.

Prolonged treatment of cells with genistein modulates the expression and function of the cystic fibrosis transmembrane conductance regulator.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. In the search for new CF therapies, small molecules have been identified that rescue the defective channel gating of CF mutants (termed CFTR potentiators). Here, we investigate the long-term effects of genistein, the best-studied CFTR potentiator, on the expression and function of CFTR. EXPERIMENTAL APPROACH: We pre-treated baby hamster kidney (BHK) cells expressing wild-type or F508del-CFTR (the most common CF mutant) with concentrations of genistein that potentiate (30 microM) or inhibit (100 microM) CFTR function for 2 or 24 h at 37 degrees C before examining CFTR maturation, expression and single-channel activity. KEY RESULTS: Using the iodide efflux technique, we found that genistein pre-treatment failed to restore function to F508del-CFTR, but altered that of wild-type CFTR. Pre-treatment of cells with genistein for 2 h had little effect on CFTR processing, whereas pre-treatment for 24 h either augmented (30 microM genistein) or impaired (100 microM genistein) CFTR maturation. Using immunocytochemistry, we found that all genistein pre-treatments increased the localization of CFTR protein to the cell surface. However, following the incubation of cells with genistein (100 microM) for 2 h, individual CFTR Cl(-) channels exhibited characteristics of channel block upon channel activation. CONCLUSIONS AND IMPLICATIONS: Genistein pre-treatment alters the maturation, cell surface expression and single-channel function of CFTR in ways distinct from its acute effects. Thus, CFTR potentiators have the potential to influence CFTR by mechanisms distinct from their effects on channel gating.

Expression of voltage-gated chloride channels in human glioma cells.

Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl- currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl- currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200-500 microm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 microm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200-500 microm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl-. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.

Heat-shock-induced protein synthesis is responsible for the switch-off of hsp70 transcription in Tetrahymena.

We had previously described that new RNA synthesis is required for expression of the heat shock protein HSP70. Here, we find that the HSP70 mRNA decreases its levels under stress conditions, heat shock (HS) or arsenite (As), and that its levels start to decline at the same time as maximal HSPs synthesis (including HSP70) occurs. This suggests that regulation of the hsp70 gene is mainly exerted at the transcriptional level. Accumulation of the HSP70 mRNA in cells stressed in presence of cycloheximide (CHX), indicates that (a) protein(s) non-existent before stress, possibly HSP70 itself (which is shown here to be relatively stable), is involved in negatively regulating hsp70 expression. Since degradation of the HSP70 mRNA is also shown to occur in cells heat-shocked under CHX, as seen from decay of its levels upon addition of actinomycin D (AMD), the protein(s) must repress hsp70 expression at the transcriptional level. Other conditions that affect normal protein synthesis, namely the translation inhibitor puromycin and the arginine-analog canavanine (shown here to be stress inducers in Tetrahymena pyriformis), also cause a delay in transcription-arrest of the HSP70 mRNA. Under severe stress conditions of HS (36 degrees C) or As (350 microM), the levels of HSP70 mRNA are higher than under mild stress conditions, however, no significant difference is seen in the pattern of HSP70 mRNA decay.

Bacterial transfer of large functional genomic DNA into human cells.

Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.

Stress response of Tetrahymena pyriformis to arsenite and heat shock: differences and similarities.

Molecular and cellular events associated with the response of Tetrahymena pyriformis to stress induced by sodium meta-arsenite, have been examined by pulse-labelling experiments. This stress agent induces the synthesis of two main groups of proteins, with molecular masses in the ranges 70-75 kDa and 25-29 kDa, together with other proteins of molecular masses 92, 83, 46, 42 (two species), 36 and 35 kDa. Comparison of the results with those of a previous study concerning the response of T. pyriformis to heat-shock, shows that the two main groups of proteins, as well as the 92-kDa and 35-kDa species, which seem to be similarly induced by both types of stress, display similar or identical peptide maps. Other stress proteins seem to be either heat-shock-specific or arsenite-specific. Studies using actinomycin D suggest that the response to arsenite is controlled mainly at the transcriptional level, for the 70-75-kDa group and 92-kDa proteins, but it seems that the other arsenite-induced proteins are subjected to transcriptional/translational control. In fact, results obtained by northern blotting, show that the mRNA coding for the 70-kDa stress protein is present only in stressed cells, whereas the 27-kDa-coding mRNA is present both in stressed and in unstressed cells. Inhibition of translation by cycloheximide has shown that heat-shock-induced-messengers are conserved under heat to be immediately translated upon removal of that inhibitor. Qualitatively similar results are obtained after prolonged treatments of T. pyriformis with arsenite and cycloheximide. The most striking difference between the responses of T. pyriformis to these two stress conditions is that arsenite does not repress normal protein synthesis so drastically as heat shock. In addition, our results suggest that some arsenite-induced messengers may be more stable than the corresponding heat-shock-induced messengers.

Crystallization and preliminary X-ray diffraction analysis of protein L-isoaspartyl O-methyltransferase from wheat germ.

Wheat-germ protein L-isoaspartyl O-methyltransferase (WPIMT) can initiate the conversion of L-isoaspartyl residues in a protein or peptide, which accumulate during the aging process in wheat-germ seeds, to normal L-aspartyl groups. The recombinant protein of WPIMT was overexpressed in Escherichia coli and purified to homogeneity. The protein was crystallized in the presence of S-adenosine-L-homocysteine using 2-methyl-2,4-pentanediol. Preliminary X-ray analysis indicated a tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 77.3, c = 152.9 A for cryofrozen crystals at 90 K. The crystals diffracted to 3.3 A and contain two molecules per asymmetric unit.

Ionic transport in tall columnar epithelial (TCE) cells obtained by nasal brushing from non-cystic fibrosis (CF) individuals.

Tall columnar epithelial (TCE) cells can be obtained by a non-invasive procedure through brushing the inferior turbinate and the adjacent lateral nasal wall. Here, we present results from the functional study of epithelial cells, thus obtained by using the patch-clamp technique. By patch-clamping the sub-apical region of TCE cells, we were able to identify at least three different groups of Cl- channels, namely: a) one with large conductance, rectifying, which was the most frequently found type of Cl- channel; b) a second type of small conductance, activated by cAMP and IBMX, in excised inside-out patches and voltage independent; c) a third type with a conductance around 25 pS, voltage independent, with a linear IV relationship, that could be observed in the excised inside-out configuration. The study of CFTR Cl- channel and its role in airway cell physiology has generally been conducted in cultured cells, most of which not polarized. This experimental work using freshly obtained TCE cells from the nasal epithelium, demonstrates that such cells may be one valid tool to study Cl- channels (most probably ORCC and CFTR Cl- channels) as a model for the lower respiratory epithelium.

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells.

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.

Cystic fibrosis patients with the 3272-26A-->G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane.

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.

Estudo das relacoes familiares de um adolescente psicotico. / Study of family relations of a psychotic adolescent

Estudo das relacoes familiares de um adolescente psicotico, atendido no Setor de Adolescentes do Instituto de Psiquiatria da UFRJ, durante um periodo de aproximadamente dois anos. Na introducao foi situado o referencial teorico do trabalho. A seguir, foi feito um relato descritivo do material clinico, que constou de uma anamnese e da evolucao do caso. Logo apos vem uma compreensao dinamica da evolucao do tratamento, na qual o grupo questiona o significado da trama delirante como uma denuncia dos conflitos familiares