Adolfo Baloira

Novel Mutations in BMPR2, ACVRL1 and KCNA5 Genes and Hemodynamic Parameters in Patients with Pulmonary Arterial Hypertension

Background Pulmonary arterial hypertension (PAH) is a rare and progressive vascular disorder characterized by increased pulmonary vascular resistance and right heart failure. The aim of this study was to analyze the Bone Morphogenetic Protein Receptor 2 (BMPR2), Activin A type II receptor like kinase 1 (ALK1/ACVRL1) and potassium voltage-gated channel, shakerrelated subfamily, member 5 (KCNA5) genes in patients with idiopathic and associated PAH. Correlation among pathogenic mutations and clinical and functional parameters was further analyzed. Methods and Results Forty one patients and fifty controls were included in this study. Analysis of BMPR2, ACVRL1 and KCNA5 genes was performed by polymerase chain reaction (PCR) and direct sequencing. Fifty one nucleotide changes were detected in these genes in 40 of the 41 patients; only 22 of these changes, which were classified as pathogenic, have been detected in 21 patients (51.2%). Ten patients (62.5%) with idiopathic PAH and 10 (40%) with associated PAH showed pathogenic mutations in some of the three genes. Several clinical and hemodynamics parameters showed significant differences between carriers and non-carriers of mutations, being more severe in carriers: mean pulmonary artery pressure (p = 0.043), pulmonary vascular resistence (p = 0.043), cardiac index (p = 0.04) and 6 minute walking test (p = 0.02). This differences remained unchanged after adjusting for PAH type (idiopathic vs non idiopathic). Conclusions Pathogenic mutations in BMPR2 gene are frequent in patients with idiopathic and associated PAH group I. Mutations in ACVRL1 and KCNA5 are less frequent. The presence of these mutations seems to increase the severity of the disease.

Mutations in the Gene Encoding Bone Morphogenetic Protein Receptor 2 in Patients With Idiopathic Pulmonary Arterial Hypertension

OBJECTIVE Pulmonary arterial hypertension (PAH) is a rare disease that can have a familial component. It has been shown that more than 50% of cases of familial PAH are associated with mutations in the gene encoding bone morphogenetic protein receptor 2 (BMPR2), which acts as a receptor for members of the transforming growth factor beta superfamily. Some studies in patients with idiopathic PAH have also shown varying percentages of mutations in this gene. The aim of this study was to determine the frequency of these mutations in a group of patients with idiopathic PAH. PATIENTS AND METHODS The study population included patients with idiopathic PAH who were seen during 2006 in our unit specialized in this entity. Patients were excluded if they had relatives who had been diagnosed with PAH or who had symptoms that led to suspicion of the disease. Diagnosis was obtained according to the protocol used in our unit. A hemodynamic study was carried out in all cases and patients were included if they had a mean pulmonary arterial pressure of greater than 25 mm Hg. DNA was extracted from peripheral leukocytes and amplified by polymerase chain reaction. Seventeen primer pairs were used for the 13 exons that make up the gene. Using the single strand conformational polymorphism (SSCP) technique we detected anomalous DNA fragments for subsequent sequencing. RESULTS The study included 8 patients (4 women). In 5 patients, no abnormalities were observed, whereas in the remaining 3, anomalous electrophoresis patterns were obtained in the SSCP and sequencing revealed mutations. In 1 case, 2 different electrophoresis patterns were observed by SSCP, but it was only possible to sequence 1 of them due to the low concentration of DNA obtained. CONCLUSIONS The presence of mutations in the gene encoding BMPR2 is not infrequent in patients with idiopathic PAH, suggesting that this family of growth factors may be important in the pathogenesis of the disease and could have therapeutic implications.

Complex inheritance in Pulmonary Arterial Hypertension patients with several mutations.

Pulmonary Arterial Hypertension (PAH) is a rare and progressive disease with low incidence and prevalence, and elevated mortality. PAH is characterized by increased mean pulmonary artery pressure. The aim of this study was to analyse patients with combined mutations in BMPR2, ACVRL1, ENG and KCNA5 genes and to establish a genotype-phenotype correlation. Major genes were analysed by polymerase chain reaction (PCR) and direct sequencing. Genotype-phenotype correlation was performed. Fifty-seven (28 idiopathic PAH, 29 associated PAH group I) were included. Several mutations in different genes, classified as pathogenic by in silico analysis, were present in 26% of PAH patients. The most commonly involved gene was BMPR2 (12 patients) followed by ENG gene (9 patients). ACVRL1 and KCNA5 genes showed very low incidence of mutations (5 and 1 patients, respectively). Genotype-phenotype correlation showed statistically significant differences for gender (p = 0.045), age at diagnosis (p = 0.035), pulmonary vascular resistance (p = 0.030), cardiac index (p = 0.035) and absence of response to treatment (p = 0.011). PAH is consequence of a heterogeneous constellation of genetic arrangements. Patients with several pathogenic mutations seem to display a more severe phenotype.

A proposal for the withdrawal of inhaled corticosteroids in the clinical practice of chronic obstructive pulmonary disease

According to the current clinical practice guidelines for chronic obstructive pulmonary disease (COPD), the addition of inhaled corticosteroids (ICS) to long-acting β2 agonist therapy is recommended in patients with moderate-to-severe disease and an increased risk of exacerbations. However, ICS are largely overprescribed in clinical practice, and most patients are unlikely to benefit from long-term ICS therapy.Evidence from recent randomized-controlled trials supports the hypothesis that ICS can be safely and effectively discontinued in patients with stable COPD and in whom ICS therapy may not be indicated, without detrimental effects on lung function, health status, or risk of exacerbations. This article summarizes the evidence supporting the discontinuation of ICS therapy, and proposes an algorithm for the implementation of ICS withdrawal in patients with COPD in clinical practice.Given the increased risk of potentially serious adverse effects and complications with ICS therapy (including pneumonia), the use of ICS should be limited to the minority of patients in whom the treatment effects outweigh the risks.

Polimorfismos en el gen de la proteína transportadora de serotonina (SERT) en pacientes con hipertensión arterial pulmonar

UNLABELLED Serotonin is a potent vasoconstrictor and pulmonary vascular growth factor whose concentration is increased in patients with pulmonary arterial hypertension (PAH). Its functions are mediated in part by the serotonin transporter protein (SERT) whose gene can have two allelic forms, both long (L) and short (S). The first was associated with greater function. OBJECTIVES To determine whether the prevalence of the L allelic form of SERT is higher in patients with PAH than in the general population. To observe whether there are any clinical differences in patients with PAH based on the SERT allele. METHODS We included patients diagnosed with PAH with catheterization based on the established criteria. Peripheral blood samples were taken and the DNA was extracted from the peripheral leukocytes. We amplified the promoter region of SERT by polymerase chain reaction and separated the products by electrophoresis. The patient samples were compared with samples from 50 healthy controls and among the most common types of PAH (idiopathic, thromboembolic and associated with connective tissue disorders). Several clinical variables were assessed according to the SERT gene alleles. RESULTS The study included 50 patients, and adequate samples were obtained in 49 (30 women). Mean age at diagnosis was 56 ± 16 years. No differences were seen in the distribution of alleles between patients and controls (P = .54). There were no differences among the three most common types of PAH (P = .3). The most frequent allelic form was LS (54% patients, 56% controls). There were no differences in either age of diagnosis or response to treatment according to the SERT alleles. There was a trend toward higher mean pulmonary pressure levels in the LL forms (49 ± 5 mm Hg vs 42 ± 9 mm Hg, P = .07). CONCLUSIONS The distribution of SERT gene alleles does not appear to be different in patients with PAH than in the normal population. Different types of PAH have a similar distribution of alleles. The LL forms do not appear to confer either clinical differences or differences in response to treatment.

Análisis de la metilación de la región promotora del gen BMPR2 en pacientes con hipertensión arterial pulmonar

INTRODUCTION Pulmonary arterial hypertension is characterizated by obstruction of the pulmonary arteries. The gene mainly related to pathology is the bone morphogenetic protein receptor type II (BMPR2). The aim of this study was to analyze the methylation pattern of the BMPR2 promoter region in patients and controls. METHODS We used Methyl Primer Express(®) v.1.0 and MatInspector softwares to analyze this region. Genomic DNA obtained from the peripheral blood of patients and controls was modified with sodium bisulphite. Methylation was analyzed using methylation-specific PCR. DNA treated with CpG methyltransferase was used as a positive control for methylation and H1299 cell culture DNA was used as positive control for gene expression. RESULTS We identified a CpG island, which may have been methylated, in the BMPR2 promoter region, in addition to NIT-2 (global-acting regulatory protein), sex-determining region Y) and heat shock factor transcription factor binding sites. We found no evidence of methylation in patients and controls. No methylated CpG sites were identified in H1299 cells expressing the BMPR2 gene. CONCLUSIONS The BMPR2 promoter region is the most suitable for study because of the high number of transcription factor binding sites that could alter gene function. No evidence of methylation was detected in this region in patients and controls.

Neumonía intersticial descamativa y bronquiolitis respiratoria asociada a enfermedad pulmonar intersticial: datos del registro español

Dentro de las neumopatias intersticiales idiopaticas, la bronquiolitis respiratoria asociada a enfermedad pulmonar intersticial (BR-EPI) y la neumonia intersticial descamativa (NID) forman un subgrupo de enfermedades raras que comparten una relacion clara con el habito tabaquico. Se han publicado pocas series con un numero importante de pacientes. En este trabajo se describen las caracteristicas de 19 casos (12 con NID y 7 con BR-EPI) recogidos en nuestro pais. Se detallan las caracteristicas clinicas, radiologicas y evolutivas, incluidos los tratamientos empleados y sus resultados. Excepto 2 pacientes con NID, todos eran o habian sido fumadores. Tos y disnea fueron los sintomas de inicio mas frecuentes, sin diferencias entre las 2 enfermedades. La alteracion radiologica predominante en la NID fue el patron en vidrio deslustrado, y en la BR-EPI, la presencia de nodulos pulmonares. La mayor parte de los pacientes recibieron tratamiento con esteroides. El pronostico en general fue bueno, y fallecio unicamente un paciente con NID.

Molecular and functional characterization of the BMPR2 gene in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is a progressive disease that causes the obstruction of precapillary pulmonary arteries and a sustained increase in pulmonary vascular resistance. The aim was to analyze functionally the variants found in the BMPR2 gene and to establish a genotype-phenotype correlation. mRNA expression studies were performed using pSPL3 vector, studies of subcellular localization were performed using pEGFP-N1 vector and luciferase assays were performed using pGL3-Basic vector. We have identified 30 variants in the BMPR2 gene in 27 of 55 patients. In 16 patients we detected pathogenic mutations. Minigene assays revealed that 6 variants (synonymous, missense) result in splicing defect. By immunofluorescence assay, we observed that 4 mutations affect the protein localization. Finally, 4 mutations located in the 5′UTR region showed a decreased transcriptional activity in luciferase assays. Genotype-phenotype correlation, revealed that patients with pathogenic mutations have a more severe phenotype (sPaP p = 0.042, 6MWT p = 0.041), a lower age at diagnosis (p = 0.040) and seemed to have worse response to phosphodiesterase-5-inhibitors (p = 0.010). Our study confirms that in vitro expression analysis is a suitable approach in order to investigate the phenotypic consequences of the nucleotide variants, especially in cases where the involved genes have a pattern of expression in tissues of difficult access.

El fenotipo no exacerbador en la enfermedad pulmonar obstructiva crónica: ¿es necesario ir un poco más allá?

Que las exacerbaciones son un aspecto fundamental de la enfermedad pulmonar obstructiva crónica (EPOC), está fuera de toda duda, y como tal aparece recogido en las diferentes guías y documentos relacionados con el manejo de esta enfermedad. Pero a partir de ahí, todo se complica. Empezando por la imprecisa definición de exacerbación y siguiendo por la subjetividad implícita en la clasificación de la gravedad (dar o no tratamiento con antibióticos o esteroides sistémicos, ingresar o no en un hospital) buena parte de las variables asociadas a las exacerbaciones se mueven entre límites muy poco precisos. Es probable que sea difícil precisar mucho más en un evento que, en bastantes casos, supone un continuum sobre los síntomas que basalmente presenta el paciente, síntomas también sujetos a variaciones relacionadas con las múltiples variables a las que cualquier individuo se expone diariamente. La guía GesEPOC nació con el objetivo de racionalizar los diferentes aspectos que incluye el manejo de un paciente con esta enfermedad y, por ello, puso especial énfasis en hacer una clasificación de los pacientes en fenotipos clínicos adaptando el tratamiento a cada uno de ellos1. Un fenotipo es el resultado de la interacción entre la carga genética y el ambiente, en el caso de la EPOC fundamentalmente la exposición al humo de cigarrillos, sin olvidar aspectos tan importantes como la nutrición, el medio sociocultural o la presencia de otros irritantes o alérgenos. La deficiencia hereditaria de alfa 1-antitripsina está claramente documentada como alteración genética que favorece una forma particular de EPOC, pero no justifica más allá del 1% de todos los casos. En los últimos años se están realizando numerosos estudios con técnicas de asociación de amplias zonas del genoma que poco a poco van identificando vías celulares cuya expresión genética posiblemente tenga gran importancia en el fenotipo clínico de la enfermedad2. La clasificación en fenotipos propuesta inicialmente por GesEPOC diferencia claramente 2 grupos, uno con tendencia a tener exacerbaciones y otro que no las tiene al menos por encima de un determinado número. En el grupo exacerbador se profundiza más en la clasificación, con 2 fenotipos diferentes, además delIt is beyond doubt that exacerbations are a fundamental aspect of chronic obstructive pulmonary disease (COPD), and this fact is reflected in the different guidelines and documents that address the management of this disease. Beyond this, however, everything becomes complicated. Starting with the imprecise definition of exacerbation, and continuing with the subjective nature of severity classifications (to treat or not to treat with antibiotics or systemic corticosteroids, to hospitalize or not to hospitalize), a hefty proportion of the variables associated with exacerbations fall within largely undefined limits. It is probably difficult to say much more about an event that, in many cases, is simply a continuum of the patient’s underlying symptoms, and these are equally susceptible to variations associated with the multiple variables to which a patient is exposed on a day-to-day basis. The GesEPOC guidelines were produced with the aim of streamlining the different aspects involved in the management of a patient with this disease, and, as such, placed particular emphasis on classifying patients by clinical phenotypes and adapting treatment accordingly.1 A phenotype is the result of the interaction between genetics and the environment

Pulmonary hypertension associated with Whipple disease

To the Editor: Whipple disease is a rare multi-systemic disorder caused by Thropheryma whipplei , a Gram-positive bacillus. Gastrointestinal manifestations are the most frequent, but many other organs may be involved [1, 2]. Pulmonary hypertension (PH) associated with Whipple disease is extremely rare, and only a few isolated cases have been reported. We present two patients with PH-Whipple disease with a positive vasodilator test and excellent response to antibiotic therapy. A 72-year-old Caucasian male with an 8-month history of intermittent abdominal pain and diarrhoea presented with dyspnoea on exertion that had progressed in the last 4 months (functional class III) and two-pillow orthopnoea to keep his head at 30°. He reported weight loss of 6 kg during the preceding 8 months. A colonoscopy performed 15 days before showed only nonspecific inflammation. Past medical history included mild chronic obstructive pulmonary disease treated with ipratropium. Physical examination revealed hypotension (systolic arterial pressure 90 mmHg) and bibasilar rales but not lower leg oedema. Laboratory data showed haemoglobin of 94 g·L−1 and albumin of 19 g·L−1. Antinuclear antibodies, rheumatoid factor and a HIV test were negative. A chest computed tomography (CT) with embolism protocol demonstrated bilateral pleural and pericardial effusions and mediastinal adenopathy. No emboli were found in the pulmonary arteries. Abdominal CT showed mild ascites and multiple small lymph nodes. Gastroduodenoscopy demonstrated diffuse inflammation in the duodenum. Small bowel biopsies showed typical periodic acid–Schiff (PAS)-positive diastase-resistant histiocytes. Several days later PCR confirmed the presence of T. whipplei . Transthoracic echocardiography showed normal left cavities with a dilated right ventricle (55 mm). The velocity of the tricuspid jet was 3.68 m·s−1 with an estimated systolic pulmonary artery pressure (sPAP) of 64 mmHg. A ventilation/perfusion scan excluded thromboembolic disease. Right heart catheterisation confirmed the diagnosis of PH (table …