Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes.

Despite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

Risk of cataract in smokers: A meta-analysis of observational studies. / Riesgo de catarata en fumadores: metaanálisis de estudios observacionales.

OBJECTIVE: The aim of the study was to compare the risk of cataract in smokers and ex-smokers. METHODS: A systematic search of observational studies was carried out in Medline, Embase, and Lilacs databases. Studies that have evaluated the association between cigarette smoking and any type of clinically diagnosed cataract were selected. The association estimators were extracted, adjusted at least by age, and were combined using random-effects models, by subtype of study (cohort, case control and cross sectional), subtype of cataract (nuclear, cortical, and posterior subcapsular), and exposure (current smoker or ex-smoker). Statistical heterogeneity, meta-regression analysis and publication bias were assessed. RESULTS: A total of 13 cohort studies, 12 case-control studies, and 18 cross-sectional studies were selected. A risk of cataract was found in current smokers: cohort (OR: 1.41; 95%CI: 1.24-1.60), cases and controls (OR: 1.45; 95%CI: 1.08-1.96), and cross-sectional studies (OR: 1.21; 95%CI: 1.09-1.34); risk of nuclear cataract: cohort (OR: 1.71; 95%CI: 1.47-1.98), case-control (OR: 1.79; 95%CI: 1.43-2.25), and cross sectional studies (OR: 1.45; 95%CI: 1.27-1.65). There was no risk of cortical or posterior subcapsular cataract in ex-smokers. CONCLUSIONS: There is a risk of cataract in smokers, particularly nuclear type. With cross-sectional studies, similar results are obtained with cohorts and cases and controls.

Evaluación radiológica de muñeca para visualizar la superficie articular del radio / Wrist X ray for analysis of the articular surface of distal radius

Resumen: En la fractura del radio distal se requieren proyecciones radiológicas que permitan ver adecuadamente la superficie articular sin interposición de estructuras. El objetivo fue determinar si las proyecciones radiológicas laterales a 7 y 22o mejoran el análisis de esta superficie. Método: Serie de casos con radiografías de pacientes sanos y operados para identificar las facetas del semilunar y escafoides en las proyecciones lateral y anteroposterior, igualmente se evaluó con tornillos. Se analizaron cualitativamente los hallazgos obtenidos en las radiografías de 7 y 22o para la proyección lateral y de 11o en la anteroposterior. Resultados: 14 radiografías de voluntarios sanos, 10 pacientes con fractura de radio que recibieron osteosíntesis y dos piezas anatómicas. En los 14 sanos y los 10 pacientes se encontró que en las proyecciones a 7 y 22o pueden apreciarse mejor las carillas radio-semilunar y radio-escafoides respectivamente, observando imágenes con menor superposición de estructuras en la radiografía de 22o tanto en los sanos como en los pacientes con fracturas. Discusión: Las proyecciones radiológicas son importantes para poder determinar los resultados inmediatos de una osteosíntesis realizada en una fractura de radio distal. En este estudio se observa que la proyección lateral a 7o identifica mejor la posición de los tornillos ubicados en la carilla semilunar del radio. La proyección lateral a 22o muestra mejor la carilla con el escafoides. Por último en la proyección anteroposterior a 11o nos permite ver la articulación radiocarpiana con menor superposición de imágenes.

Abstract: In the distal radius fracture requires radiographic views that allow you to see the articular surface without interposition. The objective was to determine whether lateral radiographic projections 7 and 22o improve the analysis of this surface. Method: Case series study with radiographs of healthy and operated patients, in order to identify the lunate and scaphoid facets in lateral and anteroposterior projections. Qualitative analysis was made on the radiographs of the distal radius with wedges of 7 and 22o in the lateral views and 11o in the anteroposterior view. Results: There were evaluated 14 radiographs of the distal radius of healthy volunteers and 10 patients with distal radius fractures who recieved surgery with internal fixation, and also two anatomical models. In 14 healthy and 10 patients, it was found that the views at 7 and 22o can be better appreciated radio lunate and radio scaphoid surface respectively, observing images with less overlapping in the radiograph of 22o in both groups. Discussion: Radiographic views are important to determine the immediate results of fixation on a distal radius fracture. We observe that the lateral view at 7o is better to show the screws on the lunate facet of the radius. The lateral view at 22o is better to show the facet of the radius with the scaphoid. Finally, the anteroposterior projection at 11o allows us to see the radio carpal joint with lower image overlay.

[Wrist X ray for analysis of the articular surface of distal radius]. / Evaluación radiológica de muñeca para visualizar la superficie articular del radio.

In the distal radius fracture requires radiographic views that allow you to see the articular surface without interposition. The objective was to determine whether lateral radiographic projections 7 and 22° improve the analysis of this surface. METHOD: Case series study with radiographs of healthy and operated patients, in order to identify the lunate and scaphoid facets in lateral and anteroposterior projections. Qualitative analysis was made on the radiographs of the distal radius with wedges of 7 and 22° in the lateral views and 11° in the anteroposterior view. RESULTS: There were evaluated 14 radiographs of the distal radius of healthy volunteers and 10 patients with distal radius fractures who recieved surgery with internal fixation, and also two anatomical models. In 14 healthy and 10 patients, it was found that the views at 7 and 22° can be better appreciated radio lunate and radio scaphoid surface respectively, observing images with less overlapping in the radiograph of 22° in both groups. DISCUSSION: Radiographic views are important to determine the immediate results of fixation on a distal radius fracture. We observe that the lateral view at 7° is better to show the screws on the lunate facet of the radius. The lateral view at 22° is better to show the facet of the radius with the scaphoid. Finally, the anteroposterior projection at 11° allows us to see the radio carpal joint with lower image overlay.

En la fractura del radio distal se requieren proyecciones radiológicas que permitan ver adecuadamente la superficie articular sin interposición de estructuras. El objetivo fue determinar si las proyecciones radiológicas laterales a 7 y 22° mejoran el análisis de esta superficie. Método: Serie de casos con radiografías de pacientes sanos y operados para identificar las facetas del semilunar y escafoides en las proyecciones lateral y anteroposterior, igualmente se evaluó con tornillos. Se analizaron cualitativamente los hallazgos obtenidos en las radiografías de 7 y 22° para la proyección lateral y de 11° en la anteroposterior. Resultados: 14 radiografías de voluntarios sanos, 10 pacientes con fractura de radio que recibieron osteosíntesis y dos piezas anatómicas. En los 14 sanos y los 10 pacientes se encontró que en las proyecciones a 7 y 22° pueden apreciarse mejor las carillas radio-semilunar y radio-escafoides respectivamente, observando imágenes con menor superposición de estructuras en la radiografía de 22° tanto en los sanos como en los pacientes con fracturas. Discusión: Las proyecciones radiológicas son importantes para poder determinar los resultados inmediatos de una osteosíntesis realizada en una fractura de radio distal. En este estudio se observa que la proyección lateral a 7° identifica mejor la posición de los tornillos ubicados en la carilla semilunar del radio. La proyección lateral a 22° muestra mejor la carilla con el escafoides. Por último en la proyección anteroposterior a 11° nos permite ver la articulación radiocarpiana con menor superposición de imágenes.

Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum.

Identification of G protein-coupled receptors and their specific function in a given neuron becomes essential to better understand the variety of signal transduction mechanisms associated with neurotransmission. We hypothesized that angiotensin II type 1 (AT1) and dopamine D2 receptors form heteromers in the central nervous system, specifically in striatum. Using bioluminescence resonance energy transfer, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors. Heteromerization did not affect cAMP signaling via D2 receptors but attenuated the coupling of AT1 receptors to Gq. A common feature of heteromers, namely cross-antagonism, i.e. the blockade of the signaling of one receptor by the blockade of the partner receptor, was tested in co-transfected cells. Candesartan, the selective AT1 receptor antagonist, was able to block D2-receptor mediated effects on cAMP levels, MAP kinase activation and ß-arrestin recruitment. This effect of candesartan, which constitutes a property for the dopamine-angiotensin receptor heteromer, was similarly occurring in primary cultures of neurons and rat striatal slices. The expression of heteromers in striatum was confirmed by robust labeling using in situ proximity ligation assays. The results indicate that AT1 receptors are expressed in striatum and form heteromers with dopamine D2 receptors that enable drugs selective for the AT1 receptor to alter the functional response of D2 receptors.

Heteromerization of GPR55 and cannabinoid CB2 receptors modulates signalling.

BACKGROUND AND PURPOSE: Heteromerization of GPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer-selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55. EXPERIMENTAL APPROACH: The direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 cells was assessed by co-immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross-talk on signalling was investigated at downstream levels by label-free real-time methods (Epic dynamic mass redistribution and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays. KEY RESULTS: GPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors. CONCLUSIONS AND IMPLICATIONS: Heteromers, unique signalling units, form in HEK293 cells expressing GPR55 and CB2 receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor.

CB1 and GPR55 receptors are co-expressed and form heteromers in rat and monkey striatum.

Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB1-GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor-receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 and GPR55 receptors form heteromers in this specific CNS region.

Valproate reduces CHOP levels and preserves oligodendrocytes and axons after spinal cord injury.

Spinal cord injury (SCI) is a major cause of disability to which there are not yet effective treatments. We previously reported that degeneration of oligodendrocytes and neurons that occurs after SCI is associated with the development of endoplasmic reticulum (ER) stress and the progressive accumulation of the pro-apoptotic factor CHOP. Since following ER stress, the balance between the pro-survival chaperone BiP and CHOP drives the cell destiny, we aimed to find drugs that modulate this ratio in favour of the former. We found that valproate (VPA) induced a significant reduction of CHOP levels after ER stress in an organotypic-based culture of spinal cord in vitro. We then administered different doses of VPA to rats following spinal cord contusion, and found that the treatment caused a marked reduction of CHOP levels early after the lesion. In addition, VPA administration partially prevented cord tissue, myelin and axonal loss, and significantly increased the relative number of surviving oligodendrocytes in the damaged spinal cord. Besides, VPA-treated rats showed better recovery of the locomotor activity than vehicle-treated rats after SCI. Since VPA is a drug already in clinical use, these results open the avenue for its therapeutical use in SCI as well as in demyelinating disorders.

[HIV retinal vasculitis]. / Vasculitis retiniana por VIH.

CASE REPORT: We present a case of a 47 year-old woman, infected with human immunodeficiency virus (HIV) diagnosed 5 years ago without receiving any treatment, who had floaters in her left eye. A peripheral retinal vasculitis was discovered and confirmed by an angiography. No source of infection was found, antiretroviral and corticosteroid treatment was given, with a complete resolution of the vasculitis. DISCUSSION: From 70-80% of positive untreated HIV patients develop ocular complications, with intraocular inflammation in more than half of them. Intraocular inflammation can be associated with opportunistic infections, tumours and as in our case, secondary to the HIV. Antiretroviral therapy is the proper treatment in these patients.

[Unilateral exophthalmos as the debut of a non-secretory multiple myeloma]. / Exoftalmos unilateral como debut de mieloma múltiple no secretor.

CASE REPORT: A 56 year-old male presented blurred vision and diplopia for 2 months, left unilateral exophthalmos, restricted ocular motility and papilledema. The imaging proofs showed osteolytic lesions in the left sphenoid bone, fourth rib and fourth dorsal vertebral body with associated masses of soft tissues. Biopsy was performed and the diagnosis of plasma cell neoplasm was established. The diagnosis of non-secretory multiple myeloma was made by analytical criteria and bone marrow biopsy. Local radiotherapy and polychemotherapy was prescribed. CONCLUSIONS: The ophthalmologist can play an important role in the diagnosis of systemic neoplasms that require the intervention of a multidisciplinary team.

[Characteristics of neonates with and without a single umbilical artery. Analysis of two consecutive series of neonates with and without congenital defects]. / Características de los neonatos con y sin arteria umbilical única. Análisis de dos series consecutivas de recién nacidos con y sin defectos congénitos.

BACKGROUND AND OBJECTIVE: The implications of the presence of a single umbilical artery (SUA) are unknown. Although most articles are based on selected samples, they suggest a relationship between SUA and malformations. Consequently, prenatal detection of SUA causes concern, since there are no definitive guidelines that can be followed after identification of this abnormality. The objective of this study was to comparatively analyze SUA in two series of consecutive births, with and without congenital defects. PATIENTS AND METHODS: A total of 19,909 cases and 19,148 controls from the Registry of the Spanish Collaborative Study on Congenital Malformations were studied. The variables analyzed were sex, birth weight, length, occipito-frontal circumference, gestational age, prematurity, delivery by caesarean section, umbilical cord length, placental weight, survival at 72 hours, primiparity, oligohydramnios, and polyhydramnios. Calculations included relative frequency, odds ratios (OR) and 95 % confidence intervals, the chi-square test, Fisher's p-value, and Student's t-test. RESULTS: SUA was found in 2.29 % of cases and in 1.03 % of controls (p = 0.0000001). These figures showed secular variation due to improvements in prenatal diagnosis and interruption of some pregnancies. When cases with and without SUA were compared, those with SUA had lower values of somatometry at birth, umbilical cord length and gestational age and had a higher risk for oligohydramnios, polyhydramnios, caesarean section, and death in the first 72 hours. Among controls, the only differences were a shorter umbilical cord and a higher frequency of oligohydramnios among infants with SUA. CONCLUSIONS: The results suggest that certain malformations associated with SUA could cause some of the differences among cases. Shortening of the umbilical cord and oligohydramnios could be related to SUA, as these abnormalities were found in both cases and controls. Comparison of cases and controls suggests that the etiopathogenesis of SUA could differ in the two groups.

Pre-gestational maternal body mass index predicts an increased risk of congenital malformations in infants of mothers with gestational diabetes.

AIMS: The aim of the present study was to identify characteristics in women diagnosed with gestational diabetes mellitus (GDM) that could be predictive of congenital malformations in their infants. METHODS: Using data from the Spanish Collaborative Study of Congenital Malformations (ECEMC), a hospital-based case-control study and surveillance system, we assessed the relationship between a number of maternal variables, including pre-gestational body mass index (BMI), and specific congenital malformations in their infants. RESULTS: The overall risk for a selected group of congenital malformations in an infant of an obese mother with GDM compared with an obese mother with normal glucose tolerance (NGT) was 2.78 (1.38-5.55, P < 0.001). Within the group of mothers with GDM, obesity (BMI > or = 30 kg/m2) was associated with a significantly increased risk of cardiovascular defects compared with non-obese women [OR = 2.82 (1.31-7.04), P < 0.01]. In mothers with NGT, pre-gestational BMI was not associated with congenital malformations. CONCLUSION: Pre-gestational obesity is a predictive variable for congenital malformations in infants of mothers with GDM. The greater their BMI, the higher the risk for congenital malformations in their offspring. Given the blastogenic origin of the congenital defects identified, and the relationship between obesity and Type 2 diabetes, it is probable that this increased risk is as a result of previously unidentified pre-gestational diabetes mellitus (PGD). It is important that overweight and obese women planning a pregnancy be evaluated for the presence of diabetes.

Pharmacokinetics of prednisolone in man during acute and chronic exposure to high altitude.

INTRODUCTION: The exposure to high altitude (H) produces several physiologic alterations which may induce changes in the pharmacokinetics of drugs. This hypothesis has been confirmed in previous studies which suggest that drugs which are highly bound to plasma proteins are most likely to exhibit altered pharmacokinetics. OBJECTIVES: To further elucidate the influence of H on pharmacokinetics, prednisolone was selected, since it is highly bound to plasma proteins, renally excreted and poorly bound to red blood cells. SUBJECTS, MATERIALS AND METHODS: Prednisolone (80 mg) was given orally to three groups of young healthy volunteers. One group was residing at sea level (L): the same volunteers were studied again after 15 hours of exposure to high altitude (3600 m, HA group), and volunteers living at H for at least six months (group HC). RESULTS: There were no statistically significant differences in the pharmacokinetic parameters calculated from plasma data in the three situations studied. When calculated from whole blood data, however, AUC and Cmax were increased and both volume of distribution and clearance diminished after exposure to H, either acute or chronically. Binding to proteins increased significantly after H exposure from 57% in group L to 75% and 94% in group H and HC, respectively. Binding to erythrocytes also increased with H exposure from 43.7% in group L to 50.6% and 61.6% in group HA and HC, respectively. The prednisolone/prednisone ratio in urine was 11.1 in group L, 7.3 in group HA and 45.6 in group HC. CONCLUSION: Since prednisone has very little intrinsic glucocorticoid activity and has to be converted to prednisolone for therapeutic effect, the alteration of the prednisolone/prednisone ratio, as a result of high altitude exposure could be clinically relevant. Additional experiments are desirable to further evaluate this observation.

Effects of high altitude exposure on the pharmacokinetics of furosemide in healthy volunteers.

INTRODUCTION: A cascade of pathophysiological events occurs with the ascension to high altitude (H). We have performed studies on the effects of exposure to H on the pharmacokinetics of drugs. The hypothesis behind these studies has been that the exposure to H, which produces marked physiological changes in the body, may alter pharmacokinetics, and consequently, pharmacodynamics. Our previous studies suggest that drugs highly bound to plasma proteins are most likely to exhibit altered disposition. OBJECTIVE: In continuation of our research, we selected furosemide which is about 98% bound to plasma proteins, renally excreted and has low binding to red blood cells. SUBJECTS, MATERIALS AND METHODS: Furosemide (40 mg) was administered orally to 3 groups of young healthy volunteers. One group who had been residing at sea level (group L), the same group after 15 hours of exposure to high altitude (3,600 m, group HA) and a group of volunteers living at H for at least 6 months (group HC). RESULTS: Our results are in accordance with the most recent pharmacokinetic studies on furosemide in which a terminal half-life of approximately 20-30 h was reported. Total proteins were 9.3% and 12.7% higher in groups HA and HC, respectively, than in group L. Albumin in group HC was 8.2% higher than group L. Bilirubin increased 17.7% and 41.2% in groups HA and HC, respectively, in comparison with group L. A rapid disposition rate constant in groups HA and HC was the only pharmacokinetic parameter that was significantly different from those in group L. Concentration of furosemide in plasma water increased significantly after H exposure, thus, the binding diminished from 97.2% in group L to 95.1% and 91.1% in groups HA and HC, respectively. CONCLUSION: Exposure to H produces an increase in the free fraction of furosemide in humans, which could be of therapeutic importance.

Epidemiological evidence that maternal diabetes does not appear to increase the risk for Down syndrome.

In 1997, Narchi and Kulaylat, studying the incidence of Down syndrome in infants of gestational diabetic mothers, concluded that maternal diabetes increases the risk for Down syndrome, but failed to control the maternal age in their analysis. Using data from the Spanish Collaborative Study of Congenital Malformations (ECEMC), we analyzed the relationship between Down syndrome and maternal diabetes mellitus, and maternal gestational diabetes, controlling the maternal age through the pair-matching analysis, stratifying by maternal age and logistic regression analysis. The analyses show that maternal age is related either to Down syndrome as well as to both types of maternal diabetes. Thus, the overall analysis could be confounded by maternal age. Once we controlled the maternal age, the risk of maternal diabetes mellitus for Down syndrome is: odds ratio (OR) = 0.92 (0.41-2.07); P = 0.83. Controlling maternal age in gestational diabetes, the risk is OR = 1.18 (0.61-2.35); P > 0.70. Based on our results, we conclude that Down syndrome is related to maternal age, but does not seem to be related to any type of maternal diabetes.

Periconceptional exposure to contraceptive pills and risk for Down syndrome.

OBJECTIVE: There are some studies which analyzed the relationship between prenatal exposure to oral contraceptives (OCs) and Down syndrome, with conflicting results even in women using OCs and conceiving at different intervals after discontinuing the use of contraceptive pills. We analyzed the risk for Down syndrome in infants of women who become pregnant while taking OC. STUDY DESIGN: We used the data from the Spanish Collaborative Study of Congenital Malformations (ECEMC). The ECEMC is a case-control study and surveillance system. For each malformed infant (case), the next non-malformed infant of the same sex born in the same hospital is selected as a control subject, from whom the collaborating physicians collected the same data as for the malformed infant. For the present study, we used two different approaches. First, the pair-matching analysis. Second, a case-control using the rest of the total of 17,183 controls from the ECEMC database with specified data on maternal use of OCs and maternal age. To control for maternal age, we used a logistic regression analysis. RESULTS: The results show an increased risk of 2.8-fold for infants with Down syndrome in women younger than 35 years of age if the mother became pregnant while she was taking OCs. We did not observe this result for women older than 34 years of age. CONCLUSION: Our results showed that the risk for Down syndrome in infants born to mothers with less than 35 years of age (as a group) who became pregnant while taking OCs is near the risk for Down syndrome of mothers with more than 34 years of age, women who are candidates for prenatal diagnosis. Thus, based on our results, one may consider the possibility of offering prenatal diagnosis for Down syndrome to young women who became pregnant while taking OCs.