Prenatal genetic diagnosis of monogenic diseases.

Prenatal genetic diagnosis of monogenic diseases is a process involving the use of a variety of molecular techniques for the molecular characterization of a potential monogenic disease in the fetus during pregnancy. Prenatal genetic diagnosis can be performed through invasive and non-invasive methods. A distinction must be made between "NIPD" (non-invasive prenatal diagnosis), which is considered to be diagnostic, from "NIPT" (non-invasive prenatal test), which is a screening test that requires subsequent confirmation by invasive methods. The different techniques currently available aim at detecting either, previously characterized pathogenic mutations in the family, the risk haplotype associated with the familial mutation, or potential pathogenic mutation(s) in a gene associated with a diagnostic suspicion. An overview is provided of relevant aspects of prenatal genetic diagnosis of monogenic diseases. The objective of this paper is to describe the main molecular techniques currently available and used in clinical practice. A description is provided of the indications, limitations and analytical recommendations regarding these techniques, and the standards governing genetic counseling. Continuous rapid advances in the clinical applications of genomics have provided increased access to comprehensive molecular characterization. Laboratories are struggling to keep in pace with technology developments.

Donor-Specific Cell-Free DNA qPCR Quantification as a Noninvasive Accurate Biomarker for Early Rejection Detection in Liver Transplantation.

(1) Background: Graft-cell-free DNA (cfDNA) in the circulation of liver transplant recipients has been proposed as a noninvasive biomarker of organ rejection. The aim of this study was to detect donor-specific cfDNA (ds-cfDNA) in the recipient's serum after either liver damage or rejection using a qPCR-based method. (2) Methods: We proposed a qPCR method based on the amplification of 10 specific insertion-deletion (InDel) polymorphisms to detect donor-specific circulating DNA diluted in the recipient cfDNA. ds-cfDNA from 67 patients was evaluated during the first month post-transplantation. (3) Results: Graft rejection in the first month post-transplantation was reported in 13 patients. Patients without liver complications showed a transitory increase in ds-cfDNA levels at transplantation. Patients with rejection showed significant differences in ds-cfDNA increase over basal levels at both the rejection time point and several days before rejection. Receiver operator characteristic (ROC) analysis showed that ds-cfDNA levels discriminated rejection, with an AUC of 0.96. Maximizing both sensitivity and specificity, a threshold cutoff of 8.6% provided an estimated positive and negative predictive value of 99% and 60%, respectively. (4) Conclusions: These results suggest that ds-cfDNA may be a useful marker of graft integrity in liver transplant patients to screen for rejection and liver damage.

Circulating Tumor Cells Enumeration from the Portal Vein for Risk Stratification in Early Pancreatic Cancer Patients.

BACKGROUND: Effective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of single circulating tumor cell (CTC) and CTC clusters from the central venous catheter (CVC) and portal blood (PV). METHODS: In total, 7 mL of PV and CVC blood from 35 patients with early pancreatic cancer were analyzed. CTC were isolated using a positive immunomagnetic selection. The detection and identification of CTC were performed by immunocytochemistry (ICC) and were analyzed by Epi-fluorescence and confocal microscopy. RESULTS: CTC and the clusters were detected both in PV and CVC. In both samples, the CTC number per cluster was higher in patients with grade three or poorly differentiated tumors (G3) than in patients with well (G1) or moderately (G2) differentiated. Patients with fewer than 185 CTC in PV exhibited a longer OS than patients with more than 185 CTC (24.5 vs. 10.0 months; p = 0.018). Similarly, patients with fewer than 15 clusters in PV showed a longer OS than patients with more than 15 clusters (19 vs. 10 months; p = 0.004). These significant correlations were not observed in CVC analyses. CONCLUSIONS: CTC presence in PV could be an important prognostic factor to predict poor prognosis in early pancreatic cancer. In addition, the number of clustered-CTC correlate to a tumor negative differentiation degree and, therefore, could be used as a diagnostic biomarker for pancreatic cancer.

Lipofuscinosis neuronal ceroidea y síndrome de Bardet-Biedl en paciente con retinosis pigmentaria / Neuronal ceroid lipofuscinosis and Bardet-Biedl syndrome in patient with retinitis pigmentosa

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Genotype-phenotype correlation of 17 cases of Pompe disease in Spanish patients and identification of 4 novel GAA variants.

BACKGROUND: Pompe disease (PD) is an autosomal recessive metabolic disorder caused by pathogenic variants in the acid α-glucosidase gene (GAA) that produces defects in the lysosomal acid α-1,4-glucosidase. We aimed to identify genetic variations and clinical features in Spanish subjects to establish genotype-phenotype correlation. METHODS: A total of 2637 samples of patients who showed symptoms or susceptible signs of PD were enrolled in this observational study. Enzymatic activity was detected by fluorometric techniques and the genetic study was carried out using Next-Generation Sequencing. RESULTS: Fourteen different variants from 17 diagnosed patients were identified, seven males and nine females with LOPD (mean age 36.07, SD 20.57, range 7-64) and a 2-day-old boy with IOPD, four genetic variants had not been described in the literature previously, including a homozygous variant. In all of them α-glucosidase activity was decreased. Muscle weakness, respiratory distress, exercise intolerance, hypotonia, dysphagia and myalgia were commonly observed in patients. CONCLUSIONS: This study report four new genetic variants that contribute to the pathogenic variants spectrum of the GAA gene. We confirm that patients in Spain have a characteristic profile of a European population, with c.-32-13T>G being the most prevalent variant. Furthermore, it was confirmed that the c.236_246delCCACACAGTGC pathogenic variant in homozygosity is associated with early disease and a worse prognosis.

Accuracy diagnosis improvement of Fabry disease from dried blood spots: Enzyme activity, lyso-Gb3 accumulation and GLA gene sequencing.

The purpose of this study was to examine the applicability of the use of samples in dried blood spot (DBS) for the definitive diagnosis of Fabry disease (FD) in males and females and to compare the diagnostic role of α-galactosidase A activity (α-Gal A), levels of lyso-Gb3 and sequencing of the GLA gene in screening patients with suspected FD. Measurement of α-Gal A activity in suspected FD patients in DBS was made followed by lyso-Gb3 determination and GLA gene sequencing. Of the 2381 subjects analyzed, FD was confirmed in 24 patients. Thirteen different variants were considered like pathogenic, five of which had not been previously described (c.143A > G; c.455A > C; c.487G > T; c.554delA; c.1045_1046insA). None of the patients with normal enzyme activity had FD confirmation. The DBS measurement of α-Gal A was more sensitive than lyso-Gb3 levels in both men and women. Definitive diagnosis of FD from a single DBS is possible, allowing samples to be easily sent from anywhere to the reference laboratory.

Hepatitis C Virus Clearance by Direct-Acting Antivirals Agents Improves Endothelial Dysfunction and Subclinical Atherosclerosis: HEPCAR Study.

INTRODUCTION: Hepatitis C virus (HCV) infection has been related to increased cardiovascular (CV) risk. The aim of this study was to analyze the impact of sustained virological response (SVR) on endothelial dysfunction and subclinical atherosclerosis in patients with hepatitis C virus treated with direct-acting antiviral agents. METHODS: A total of 114 patients were prospectively recruited and underwent CV risk assessment including (i) endothelial dysfunction determined through laser Doppler flowmetry and (ii) subclinical atherosclerosis, elucidated by the ankle-brachial index (ABI). Atherogenic lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides); markers of oxidative stress (oxidized low-density lipoprotein antibodies [OLAbs]), soluble markers of adhesion (vascular cell adhesion molecule [VCAM], e-selectin, and soluble markers of angiogenesis; and vascular endothelial growth factor, endothelial [EMPs] and platelet [PMPs] apoptotic microparticles, and cell-free DNA [cfDNA]) were measured. All determinations were performed at baseline, 12 weeks (SVR time), and 1 year after treatment. RESULTS: In patients with endothelial dysfunction, area of hyperemia improved after virus clearance (P = 0.013) and was related to significant decrease in VCAM, e-selectin (P < 0.001), and cfDNA (P = 0.017) and to increased OLAb levels (P = 0.001). In patients with subclinical atherosclerosis at baseline, a significantly improved ABI was seen after HCV clearance (P < 0.001). Levels of both EMPs and PMPs also decreased after SVR and at follow-up (P = 0.006 and P = 0.002, respectively). DISCUSSION: HCV clearance improved not only liver function but also endothelial dysfunction and subclinical atherosclerosis promoted by decrease in levels of VCAM, e-selectin, cfDNA, and PMPs and EMPs.

Genotype/phenotype relationship in Gaucher disease patients. Novel mutation in glucocerebrosidase gene.

Objectives Gaucher disease (GD) is the most common inherited lysosomal storage disease, caused by mutations in acid ß-glucosidase (GBA) gene. This study aimed to identify mutations in Andalusia patients with GD and their genotype-phenotype correlation. Methods Descriptive observational study. University Hospital Virgen del Rocio patients diagnosed from GD from 1999 to 2019 were included. Demographic and clinical data, ß-glucocerebrosidase activity, variants pathogenic in GBA gene and biomarkers for monitoring treatment were collected from digital medical record. Results Twenty-six patients with aged between 1 day and 52 years were studied. A total of six mutations described as pathogenic and one mutation not described above [c.937T>C (p.Tyr313His)] were identified in the GBA gene, four patients were homozygotes and 22 compound heterozygotes. Twenty-four patients were diagnosed in non-neuropathic form (type 1) and two cases presented neurological involvement (type 2 or 3). The most common variant was c.1226A>G (p.Asn409Ser), which was detected in 24 patients, followed by c.1448T>C (p.Leu483Pro) variant, identified in 13 patients. The c.1448T>C (p.Leu483Pro) mutation has been presented in the most severe phenotypes with neurological involvement associated with type 2 and 3 GD, while c.1226A>G (p.Asn409Ser) mutation has not been associated with neurological alterations. Splenomegaly and bone disease were the most frequent clinical manifestations, and thrombocytopenia was the most common hematological disorder. Conclusions The c.1226A>G (p.Asn409Ser) and c.1448T>C (p.Leu483Pro) mutations were the most common. The c.937T>C (p.Tyr313His) was identified as a novel mutation. The c.1448T>C (p.Leu483Pro) mutation was associated with neurological alterations and c.1226A>G (p.Asn409Ser) mutation has not been associated it.

Family study of a novel mutation of mucopolysaccharidosis type VI with a severe phenotype and good response to enzymatic replacement therapy: Case report.

RATIONALE: Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome is produced by the deficiency of the enzyme arylsulfatase B, responsible for the hydrolysis of N-acetyl-D-galactosamine, chondroitin sulfate, and dermatan sulfate. PATIENT CONCERNS: A 3-year-old male with Moroccan origins is the index case. He had healthy consanguineous parents and 4 healthy brothers and sisters. The patient showed a wide spectrum of symptoms including skeletal dysplasia and short stature with elevated glycosaminoglycans (GAGs) in urine. DIAGNOSES, INTERVENTIONS, AND OUTCOMES: GAGs were quantified by spectrometry method with 1,9-dimethylen blue in 24-hour urine samples. The qualitative analysis of urine GAGs was obtained by thin-layer chromatography to determine the predominant presence of dermatan sulfate. The activities of both arylsulfatase B and beta-galactosidase as well as genetic studies were performed in dried blood spots. The genetic study was performed with deoxyribonucleic acid by massive sequencing a of lisosomal storage diseases. Results showed a new mutation c.263A > C with the severe phenotype in homozygous in the patient. The familiar study of ARSB and GLB1 genes presented some asymptomatic SNPs but with a discrete decrease in the activity of arylsulfatase B and beta-galactosidase. After an early detection by pediatricians, and both enzymatic and genetic confirmation, the patient had a good response to substitutive enzymatic treatment with galsulfase. LESSONS: Mucoplysaccharidosis type VI is an autosomal recessive rare disease characterized by a lysosomal storage disorder. Although a number of mutations have been already associated to the disease, we have found a new mutation located in the arylsulfatase B enzyme gene. We have described that this mutation is the ultimate cause of a severe presentation of the disease.

Noninvasive prenatal diagnosis by cell-free DNA screening for fetomaternal HPA-1a platelet incompatibility.

BACKGROUND: The development of new noninvasive approaches for the diagnosis of human platelet antigen (HPA)-1 fetomaternal incompatibility has become of great interest. These approaches allow determination of whether the fetus is incompatible or not with the mother and a decision on antenatal therapy to avoid fetal or neonatal alloimmune thrombocytopenia (FNAIT). The objective of this work was to perform rapid, noninvasive prenatal test for HPA-1ab fetal antigen detection after the detection of an HPA-1-homozygous mother by using plasma cell-free DNA (cfDNA). STUDY DESIGN AND METHODS: The HPA-1 genotypes of 142 pregnant women and 17 nonpregnant controls were retrospectively determined by high-resolution melting (HRM) polymerase chain reaction (PCR). Coamplification at lower denaturation temperature (COLD) HRM PCR was performed to determine the fetal genotype analyzing cfDNA from all HPA-1bb pregnant women. RESULTS: After the HRM analysis, the following genotypes were identified: HPA-1aa (71.13%), HPA-1bb (2.8%), and HPA-1ab (26.06%). Four HPA-1bb-homozygous pregnant women were carrying an incompatible fetus. Plasma samples from these mothers were analyzed by HRM COLD-PCR. Homozygous HPA-1bb pregnant women carrying an HPA-1ab-heterozygous fetus did not group with either the HPA-1ab or the HPA-1bb controls. Thus, COLD-PCR analysis allows the detection of HPA-1ab-heterozygous fetuses carried by homozygous mothers during first weeks of pregnancy. CONCLUSION: The fetal genotype from HPA-1bb-homozygous women was detected by a noninvasive prenatal test as soon as 12 weeks of gestation.

Consumption of orange fermented beverage improves antioxidant status and reduces peroxidation lipid and inflammatory markers in healthy humans.

BACKGROUND: Alcoholic fermentation of fruits has generated novel products with high concentrations of bioactive compounds and moderate alcohol content. The aim of this study was to evaluate the potential effect on cardiovascular risk factors of the regular consumption by healthy humans of a beverage obtained by alcoholic fermentation and pasteurization of orange juice. RESULTS: Thirty healthy volunteers were enrolled in a randomized controlled study. The experimental group (n = 15) drank 500 mL orange beverage (OB) per day for 2 weeks (intervention phase), followed by a 3-week washout phase. Blood samples were collected at baseline (E-T0) and at the end of the intervention (E-T1) and washout (E-T2) phases. Controls (n = 15) did not consume OB during a 2-week period. OB intake significantly increased oxygen radical absorbance capacity (43.9%) and reduced uric acid (-8.9%), catalase (CAT) (-23.2%), thiobarbituric acid reactive substances (TBARS) (-30.2%) and C-reactive protein (-2.1%) (E-T1 vs. E-T0). These effects may represent longer-term benefits, given the decreased uric acid (-8.9%), CAT (-34.6%), TBARS (-48.4%) and oxidized low-density lipoprotein (-23.9%) values recorded after the washout phase (E-T2 vs. E-T0). CONCLUSION: The regular consumption of OB improved antioxidant status and decreased inflammation state, lipid peroxidation and uric acid levels. Thus OB may protect the cardiovascular system in healthy humans and be considered a novel functional beverage. © 2017 Society of Chemical Industry.

Total and Fetal Circulating Cell-Free DNA, Angiogenic, and Antiangiogenic Factors in Preeclampsia and HELLP Syndrome.

BACKGROUND: Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by hypertension and proteinuria. The HELLP syndrome is the most severe form of PE. The aim of the present study was to determine different potential biomarkers that may help us perform an early diagnosis of the disease, assess on the severity of the disease, and/or predict maternal or fetal adverse outcomes. METHODS: We measured serum levels of total and fetal circulating cell-free DNA (cfDNA), soluble endoglin, soluble form of vascular endothelial growth factor receptor, and placental growth factor in a healthy control group of pregnant women (n = 26), patients with mild (n = 37) and severe PE (n = 25), and patients with HELLP syndrome (n = 16). RESULTS: We observed a gradual and strong relationship between all the biomarkers mentioned and the range of severity of PE, with the highest levels in patients with HELLP syndrome. Nevertheless, only the values of total cfDNA were able to significantly differentiate severe PE and HELLP syndrome (20957 ± 2784 vs. 43184 ± 8647 GE/ml, P = 0.01). Receiver operating characteristic (ROC) curves were constructed (i) for the healthy group with respect to the groups with PE and (ii) for patients with PE with respect to the group with HELLP syndrome; sensitivity and specificity values at different cutoff levels were calculated in each case. The maximum ROC area under the curve value for PE and HELLP syndrome (with respect to controls) was 0.91 (P < 0.001). CONCLUSIONS: The measured biomarkers of cell damage, angiogenesis, and antiangiogenesis may reflect the severity of PE, with higher levels in patients who develop HELLP syndrome. In addition, these biomarkers may also help predict adverse fetal and maternal outcomes.

Detection of p53 Mutations in Circulating DNA of Transplanted Hepatocellular Carcinoma Patients as a Biomarker of Tumor Recurrence.

p53 is the most commonly mutated gene in malignant human cancers. To detect p53 mutations in circulating DNA (cirDNA) of transplanted hepatocellular carcinoma (HCC) patients could be an interesting approach to know of any tumor recurrence. In this study, our objective was to determine the utility of this method in the diagnosis and the prognosis of HCC tumor recurrence.Twenty four liver transplanted HCC patients were included in the study together with a group of healthy controls. Detection of the specific p53 mutation in cirDNA was performed by high-resolution melting PCR (HRM-PCR) and COLD-PCR immediately before the transplantation. Serum anti-p53 was also determined using a p53-autoantibody ELISA kit.The results of the HRM-PCR and COLD-PCR showed two well-differentiated groups of transplanted patients after normalization by healthy controls. These data allow us to distinguish between patients with p53 mutated cirDNA and those with wild type cirDNA. Moreover, we have found that most of p53 mutated patients also presented elevated anti-p53 antibodies. The present results indicate that it is possible to detect mutated p53 genes with the cirDNA and that this could be used as a biomarker of tumor recurrence during the clinical evolution of the transplanted patients.

Screening of KRAS Mutation in Pre- and Post-Surgery Serum of Patients Suffering from Colon Cancer by COLD-PCR HRM.

Genomic characterization of cell-free circulating tumour DNA (ctDNA) may offer an opportunity to assess clonal dynamics throughout the course of a patient's illness. The existence of KRAS driver mutations in colon cancer patients is determinant to decide their treatment and to predict their outcome. DNA is extracted automatically from 400 µL of serum using the MagNa Pure Compact with the Nucleic Acid Isolation Kit I. DNA amplification, COLD-PCR and HRM were performed in the same run in the Light Cycler 480.We found three different situations: pre- and post-surgical samples grouped with the negative control, pre-surgical samples appear to group with the positive control and the post-surgical samples appear to group with the negative control and finally both samples, pre- and post-surgical ones, appear to be grouped with the positive control. COLD-PCR HRM is a cost-effective way for screening one of the most common driver mutations to predict the worst prognosis in colorectal cancer.

Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis.

Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.

Evaluation of the State of Transplanted Liver Health by Monitoring of Organ-Specific Genomic Marker in Circulating DNA from Receptor.

The evaluation of the transplanted liver health by non-invasive approaches may offer an improvement in early clinical intervention. As transplanted organs have genomes that are distinct from the host's genome, the quantification of the specific DNA of the donated liver in the patient serum will allow us to obtain information about its damage. We evaluated the state of transplanted liver health by monitoring the RH gene in serum circulating DNA (cirDNA) from 17 recipient and donor mismatched for this gene. cirDNA RH gene was quantified by RT- PCR before, at the moment of transplantation (day 0) and during the stay at the intensive care unit. Beta-globin cirDNA was quantified as a general cellular damage marker. Patients were grouped based on clinical outcomes: (A) patients with no complication; (B) patients that accepted the organ but suffered other complications; (C) patients that suffered organ rejection. All patients showed an increased cirDNA levels at day 0 that decreased until patient stabilization. Patients from groups A and B showed low levels of the RH gene cDNA during the follow-up, with an increase of beta-globin gene at the moment of any clinical complication. Patients from group C showed an increase in the RH gene during rejection.

Circulating cell-free DNA is a predictor of short-term neurological outcome in stroke patients treated with intravenous thrombolysis.

Circulating cell-free DNA (cfDNA) has been described as a prognostic marker for several diseases. Its prognostic value for short-term outcome in stroke patients treated with intravenous thrombolysis remains unexplored. cfDNA was measured on admission in 54 tissue plasminogen activator (tPA)-treated patients and 15 healthy controls using a real-time quantitative polymerase chain reaction assay. Neurological outcome was assessed at 48 h. Predictors of neurological improvement were evaluated by logistic regression analysis, and the additional predictive value of cfDNA over clinical variables was determined by integrated discrimination improvement (IDI). Stroke patients presented higher baseline cfDNA than healthy controls (408.5 (179-700.5) vs. 153.5 (66.9-700.5) kilogenome-equivalents/L, p = 0.123). A trend towards lower cfDNA levels was found in patients who neurologically improved at 48 h (269.5 (143.3-680) vs. 504 (345.9-792.3) kilogenome-equivalents/L, p = 0.130). In logistic regression analysis, recanalization at 1 h and cfDNA < 302.75 kilogenome-equivalents/L was independently associated with neurological improvement after adjustment by age, gender and baseline National Institutes of Health Stroke Scale score. The addition of cfDNA to the clinical predictive model improved its discrimination (IDI = 21.2% (9.2-33.3%), p = 0.009). These data suggest that cfDNA could be a surrogate marker for monitoring tPA efficacy by the prediction of short-term neurological outcome.

Pleural fluid cell-free DNA in parapneumonic pleural effusion.

OBJECTIVES: To measure the accuracy of pleural fluid cell-free DNA (cfDNA) concentration for diagnosis of parapneumonic pleural effusions (PPE). DESIGN AND METHODS: We studied pleural fluids obtained by thoracocentesis in patients with pleural effusion. DNA was automatically extracted from pleural fluid using the MagNa Pure Compact instrument (Roche Diagnostics), and was measured by a real-time quantitative PCR assay for the ß-globin gene using a Light-Cycler 480 Real-Time PCR instrument (Roche Diagnostics). Patients were classified into two groups according to the etiology of pleural effusion: PPE and NOT PPE. The diagnostic accuracy was determined using receiver operating characteristic (ROC) techniques by analyzing the area under the ROC curve (AUC). RESULTS: We studied 78 patients with ages between 1 and 86 years old (median=64). Sixteen patients were PPE and 62 were NOT PPE (24 transudative, 30 malignant and 8 other etiology). Pleural fluid cfDNA concentration was higher in patients with PPE (median=46,240 ng/mL) than in those with NOT PPE (median=224 ng/mL). The AUC value was 0.907 (p<0.0001) and the optimal cut-off value was 6740 ng/mL exhibiting 87.5% sensitivity and 80.6% specificity. Also, there were significant differences between transudative and exudative effusions according to pleural fluid cfDNA concentration (p<0.0001). The AUC value was 0.994 and the optimal cut-off value was 162ng/mL exhibiting 100% sensitivity and 96.3% specificity. CONCLUSIONS: Pleural fluid cfDNA concentration showed high accuracy for diagnosis of PPE and to discriminate between transudative and exudative effusions.

Obstructive sleep apnoea syndrome, endothelial function and markers of endothelialization. Changes after CPAP.

STUDY OBJECTIVES: This study tries to assess the endothelial function in vivo using flow-mediated dilatation (FMD) and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA) syndrome at baseline and after three months with CPAP therapy. DESIGN: Observational study, before and after CPAP therapy. SETTING AND PATIENTS: We studied 30 patients with apnoea/hypopnoea index (AHI) >15/h that were compared with themselves after three months of CPAP therapy. FMD was assessed non-invasively in vivo using the Laser-Doppler flowmetry. Circulating cell-free DNA (cf-DNA) and microparticles (MPs) were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF) was determined as a marker of endothelial restoration process. MEASUREMENTS AND RESULTS: After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, p< 0.005) cf-DNA and MPs significantly decreased (187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p<0.01, and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/µL, p<0.05, respectively) and VEGF levels increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p<0.05). These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p<0.005) but not between markers of damage and restoration, thus suggesting that both types of markers should be measured together. CONCLUSIONS: CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage.