The microbiota drives diurnal rhythms in tryptophan metabolism in the stressed gut.

Chronic stress disrupts microbiota-gut-brain axis function and is associated with altered tryptophan metabolism, impaired gut barrier function, and disrupted diurnal rhythms. However, little is known about the effects of acute stress on the gut and how it is influenced by diurnal physiology. Here, we used germ-free and antibiotic-depleted mice to understand how microbiota-dependent oscillations in tryptophan metabolism would alter gut barrier function at baseline and in response to an acute stressor. Cecal metabolomics identified tryptophan metabolism as most responsive to a 15-min acute stressor, while shotgun metagenomics revealed that most bacterial species exhibiting rhythmicity metabolize tryptophan. Our findings highlight that the gastrointestinal response to acute stress is dependent on the time of day and the microbiome, with a signature of stress-induced functional alterations in the ileum and altered tryptophan metabolism in the colon.

Succinate-Directed Approaches for Warburg Effect-Targeted Cancer Management, an Alternative to Current Treatments?

Approximately a century ago, Otto Warburg discovered that cancer cells use a fermentative rather than oxidative metabolism even though the former is more inefficient in terms of energy production per molecule of glucose. Cancer cells increase the use of this fermentative metabolism even in the presence of oxygen, and this process is called aerobic glycolysis or the Warburg effect. This alternative metabolism is mainly characterized by higher glycolytic rates, which allow cancer cells to obtain higher amounts of total ATP, and the production of lactate, but there are also an activation of protumoral signaling pathways and the generation of molecules that favor cancer progression. One of these molecules is succinate, a Krebs cycle intermediate whose concentration is increased in cancer and which is considered an oncometabolite. Several protumoral actions have been associated to succinate and its role in several cancer types has been already described. Despite playing a major role in metabolism and cancer, so far, the potential of succinate as a target in cancer prevention and treatment has remained mostly unexplored, as most previous Warburg-directed anticancer strategies have focused on other intermediates. In this review, we aim to summarize succinate's protumoral functions and discuss the use of succinate expression regulators as a potential cancer therapy strategy.

Eligibility criteria for Menopausal Hormone Therapy (MHT): a position statement from a consortium of scientific societies for the use of MHT in women with medical conditions. MHT Eligibility Criteria Group.

This project aims to develop eligibility criteria for menopausal hormone therapy (MHT). The tool should be similar to those already established for contraception A consortium of scientific societies coordinated by the Spanish Menopause Society met to formulate recommendations for the use of MHT by women with medical conditions based on the best available evidence. The project was developed in two phases. As a first step, we conducted 14 systematic reviews and 32 metanalyses on the safety of MHT (in nine areas: age, time of menopause onset, treatment duration, women with thrombotic risk, women with a personal history of cardiovascular disease, women with metabolic syndrome, women with gastrointestinal diseases, survivors of breast cancer or of other cancers, and women who smoke) and on the most relevant pharmacological interactions with MHT. These systematic reviews and metanalyses helped inform a structured process in which a panel of experts defined the eligibility criteria according to a specific framework, which facilitated the discussion and development process. To unify the proposal, the following eligibility criteria have been defined in accordance with the WHO international nomenclature for the different alternatives for MHT (category 1, no restriction on the use of MHT; category 2, the benefits outweigh the risks; category 3, the risks generally outweigh the benefits; category 4, MHT should not be used). Quality was classified as high, moderate, low or very low, based on several factors (including risk of bias, inaccuracy, inconsistency, lack of directionality and publication bias). When no direct evidence was identified, but plausibility, clinical experience or indirect evidence were available, "Expert opinion" was categorized. For the first time, a set of eligibility criteria, based on clinical evidence and developed according to the most rigorous methodological tools, has been defined. This will provide health professionals with a powerful decision-making tool that can be used to manage menopausal symptoms.

Activated prothrombin complex concentrate to treat bleeding events in acquired hemophilia A: BAHAS study.

OBJECTIVE: Activated prothrombin complex concentrate (aPCC) is a bypassing agent indicated to treat bleeds in patients with acquired hemophilia A (AHA). Nevertheless, its efficacy and safety in the real-world setting have not often been addressed. METHODS: We report the experience of Spanish reference centers for coagulation disorders and from acquired hemophilia Spanish Registry (AHASR) from August 2012 to February 2021. Follow-up period of 30 days after aPCC withdrawal. RESULTS: Thirty patients with a median age of 70 years old, suffering from 51 bleeds treated with aPCC were finally evaluated. As first-line treatment, aPCC stopped bleeding in 13 of 14 (92.9%) cases. aPCC as the second line after recombinant factor VIIa failure, stopped bleeding in all cases. In 17 patients, aPCC was used far from initial bleed control as prophylaxis of rebleeding with 94% effectiveness. No thromboembolic episodes were communicated. One patient developed hypofibrinogenemia, which did not prevent aPCC from halting bleeding. No other serious adverse events possibly or probably associated with aPCC were reported. CONCLUSIONS: This data support aPCC as hemostatic treatment in AHA with high effectiveness and excellent safety profile in acute bleeds and as extended use to prevent rebleedings, even in aging people with high cardiovascular risk.

Adrenomedullin: Not Just Another Gastrointestinal Peptide.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two bioactive peptides derived from the same precursor with several biological functions including vasodilation, angiogenesis, or anti-inflammation, among others. AM and PAMP are widely expressed throughout the gastrointestinal (GI) tract where they behave as GI hormones, regulating numerous physiological processes such as gastric emptying, gastric acid release, insulin secretion, bowel movements, or intestinal barrier function. Furthermore, it has been recently demonstrated that AM/PAMP have an impact on gut microbiome composition, inhibiting the growth of bacteria related with disease and increasing the number of beneficial bacteria such as Lactobacillus or Bifidobacterium. Due to their wide functions in the GI tract, AM and PAMP are involved in several digestive pathologies such as peptic ulcer, diabetes, colon cancer, or inflammatory bowel disease (IBD). AM is a key protective factor in IBD onset and development, as it regulates cytokine production in the intestinal mucosa, improves vascular and lymphatic regeneration and function and mucosal epithelial repair, and promotes a beneficial gut microbiome composition. AM and PAMP are relevant GI hormones that can be targeted to develop novel therapeutic agents for IBD, other GI disorders, or microbiome-related pathologies.

Type 2N VWD: Conclusions from the Spanish PCM-EVW-ES project.

INTRODUCTION: Type 2N von Willebrand disease (VWD) is characterized by a decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). Abnormal binding of FVIII to VWF (VWF:FVIIIB), results in low FVIII plasma levels, which can lead to a misdiagnosis of mild haemophilia A. Accurate diagnosis of type 2N VWD is essential for appropriate genetic counselling and therapy. This disease can be distinguished from haemophilia A by in vitro assays (measurement VWF:FVIIIB activity) and/or genetic analysis. AIM: To identify the current challenges in the diagnosis and treatment of this type of VWD and provide an in-depth description of the phenotypes and mutations identified. RESULTS: Twenty-eight patients had at least one type 2N mutation, and 13 of these had a type 2N mutation combined with other variations. Three type 2N mutations were detected: p.Arg816Trp, p.Arg854Gln, and p.Arg763Ser. Two of these are the most frequently described mutations worldwide. This mutational spectrum differs from the broad spectrum seen in neighbouring France, where at least eight distinct 2N mutations have been found. In the PCM-EVW-ES cohort, 11 asymptomatic type 2N carriers with borderline FVIII plasma levels would probably have been excluded if the evaluation had been based on clinical and laboratory data only. Likewise, three patients with a severe phenotype would have been classified as homozygous for a 2N mutation if only the phenotype study had been performed. CONCLUSION: The high detection yield and affordability of next-generation sequencing support the use of this technology as a first-line diagnostic tool in this setting.

Unraveling the Influence of Common von Willebrand factor variants on von Willebrand Disease Phenotype: An Exploratory Study on the Molecular and Clinical Profile of von Willebrand Disease in Spain Cohort.

The clinical diagnosis of von Willebrand disease (VWD), particularly type 1, can be complex because several genetic and environmental factors affect von Willebrand factor (VWF) plasma levels. An estimated 60% of the phenotypic variation is attributable to hereditary factors, with the ABO blood group locus being the most influential. However, recent studies provide strong evidence that nonsynonymous single nucleotide variants (SNVs) contribute to VWF and factor VIII phenotypic variability in healthy individuals. This study aims to investigate the role of common VWF SNVs on VWD phenotype by analyzing data from 219 unrelated patients included in the "Molecular and Clinical Profile of von Willebrand Disease in Spain project." To that end, generalized linear mixed-effects regression models were fitted, and additive and epistatic analyses, and haplotype studies were performed, considering five VWD-related measures (bleeding score, VWF:Ag, VWF:RCo, factor VIII:C, and VWF:CB). According to these analyses, homozygotes: for p.Thr789Ala(C) would be expected to show 39% higher VWF:Ag levels; p.Thr1381Ala(C), 27% lower VWF:Ag levels; and p.Gln852Arg(C), 52% lower VWF:RCo levels. Homozygotes for both p.Thr789Ala(C) and p.Gln852Arg(T) were predicted to show 185% higher VWF:CB activity, and carriers of two copies of the p.Thr1381Ala(T)/p.Gln852Arg(T) haplotype would present a 100% increase in VWF:RCo activity. These results indicate a substantial effect of common VWF variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on VWF, these findings provide new evidence regarding the contribution of common variants to VWD, which should be taken into account to enhance the accuracy of the diagnosis and classification of this condition. ClinicalTrials.gov identifier: NCT02869074.

Engineering Cercospora disease resistance via expression of Cercospora nicotianae cercosporin-resistance genes and silencing of cercosporin production in tobacco.

Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.

Identification of 58 Mutations (26 Novel) in 94 of 109 Symptomatic Spanish Probands with Protein C Deficiency.

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene (PROC) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range.

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project.

The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study 'Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)' has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.

Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer.

To investigate the contribution of adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we studied the effects of four small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. For each SM, four experimental groups of male mice were used: (i) Control group; (ii) SM group; (iii) DSS group (injected with azoxymethane [AOM] and drank dextran sulfate sodium [DSS]); and (iv) DSS + SM group (treated with AOM, DSS, and the SM). None of the mice in groups i and ii developed tumors, whereas all mice in groups iii and iv developed colon neoplasias. No significant differences were found among mice treated with PAMP modulators (87877 and 106221). Mice that received the AM negative modulator, 16311, had worse colitis symptoms than their control counterparts, whereas mice injected with the AM positive modulator, 145425, had a lower number of tumors than their controls. SM 145425 regulated the expression of proliferation marker Lgr5 and had an impact on microbiota, preventing the DSS-elicited increase of the Bacteroides/Prevotella ratio. These results suggest that treatment with AM or with positive modulator SMs may represent a novel strategy for colon cancer.

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): comprehensive genetic analysis by next-generation sequencing of 480 patients.

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

Lack of Adrenomedullin Aggravates Acute TNBS-Induced Colitis Symptoms in Mice, Especially in Females.

Adrenomedullin (AM) is a biologically active peptide which has been tested as a new therapy for inflammatory bowel disease (IBD) in animal models and in patients with severe ulcerative colitis. We used an inducible knockout (KO) mouse model for AM to evaluate the effects of endogenous levels of this peptide on the development and degree of pathogenesis of IBD. Acute colitis was induced in mice of both sexes by rectal instillation of 3 mg 2,4,6-trinitrobenzenesulfonic acid (TNBS) in 100 µL of 50% ethanol. Control mice received the same volume of saline in 50% ethanol. During the following 5 days, the weight and the disease severity index of all animals were recorded. After sacrifice, the inflammatory response was macroscopically assessed by analyzing the weight of the colon; by histomorphometrical analysis on histological sections; and by qRT-PCR determination of different inflammatory, adhesion, and regeneration molecules. TNBS administration caused a significantly more severe colitis in KO mice, and especially in females, when compared to wild type (WT) animals. Abrogation of the AM gene caused more severe diarrhea, accompanied by rectal bleeding, anorexia, and a significant increase of colon weight. Histological analysis of TNBS-treated KO mice showed large areas of lymphocyte infiltrates in the mucosa and submucosa, with loss of tissue architecture. No alterations were observed in the expression levels of inflammatory cytokines at the time of sacrifice; meanwhile lack of AM resulted in lower levels of some adhesion molecules and regeneration markers. Taken together, these results support the protective role of endogenous AM against the development of acute colitis, and that its effects are particularly beneficial on females.

Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

Lack of Adrenomedullin Results in Microbiota Changes and Aggravates Azoxymethane and Dextran Sulfate Sodium-Induced Colitis in Mice.

The link between intestinal inflammation, microbiota, and colorectal cancer is intriguing and the potential underlying mechanisms remain unknown. Here we evaluate the influence of adrenomedullin (AM) in microbiota composition and its impact on colitis with an inducible knockout (KO) mouse model for AM. Microbiota composition was analyzed in KO and wild type (WT) mice by massive sequencing. Colitis was induced in mice by administration of azoxymethane (AOM) followed by dextran sulfate sodium (DSS) in the drinking water. Colitis was evaluated using a clinical symptoms index, histopathological analyses, and qRT-PCR. Abrogation of the adm gene in the whole body was confirmed by PCR and qRT-PCR. KO mice exhibit significant changes in colonic microbiota: higher proportion of δ-Proteobacteria class; of Coriobacteriales order; and of other families and genera was observed in KO feces. Meanwhile these mice had a lower proportion of beneficial bacteria, such as Lactobacillus gasseri and Bifidobacterium choerinum. TLR4 gene expression was higher (p < 0.05) in KO animals. AM deficient mice treated with DSS exhibited a significantly worse colitis with profound weight loss, severe diarrhea, rectal bleeding, colonic inflammation, edema, infiltration, crypt destruction, and higher levels of pro-inflammatory cytokines. No changes were observed in the expression levels of adhesion molecules. In conclusion, we have shown that lack of AM leads to changes in gut microbiota population and in a worsening of colitis conditions, suggesting that endogenous AM is a protective mediator in this pathology.

Prevention of Bone Loss in a Model of Postmenopausal Osteoporosis through Adrenomedullin Inhibition.

Despite recent advances in the understanding and treatment options for osteoporosis, this condition remains a serious public health issue. Adrenomedullin (AM) is a regulatory peptide with reported activity on bone remodeling. To better understand this relationship we built an inducible knockout for AM. An outstanding feature of knockout mice is their heavier weight due, in part, to the presence of denser bones. The femur of knockout animals was denser, had more trabeculae, and a thicker growth plate than wild type littermates. The endocrine influence of AM on bone seems to be elicited through an indirect mechanism involving, at least, the regulation of insulin, glucose, ghrelin, and calcitonin gene-related peptide (CGRP). To confirm the data we performed a pharmacological approach using the AM inhibitor 16311 in a mouse model of osteoporosis. Ovariectomized females showed significant bone mass loss, whereas ovariectomized females treated with 16311 had similar bone density to sham operated females. In conclusion, we propose the use of AM inhibitors for the treatment of osteoporosis and other conditions leading to the loss of bone mass.

Transbronchial Cryobiopsy in Interstitial Lung Disease: Are We on the Right Path?

BACKGROUND: A definitive and specific diagnosis of interstitial lung disease (ILD) often requires a histologic analysis of the lung parenchyma. A transbronchial biopsy with forceps has a limited diagnostic yield for idiopathic interstitial pneumonias. The incorporation of a transbronchial lung cryobiopsy for diagnosing ILD appears to be very promising, although there are only a few published studies in this regard. Our paper shows the results of using this technique in our center. METHODS: This was a prospective study including 55 patients with ILD diagnosed from January 2012 to January 2015. The methodology used, the overall diagnostic yield, and the number and the location of samples, in addition to complications, have been reported. RESULTS: In 38 (69%), 10 (20.8%), and 7 (12.7%) cases, we obtained a certain, highly likely, and unclassifiable diagnosis of interstitial pneumonia, respectively. With 18 cases, usual interstitial pneumonia was the most common diagnosis. The most common complication was a pneumothorax in 8 patients (14.5%). CONCLUSIONS: A transbronchial lung cryobiopsy using a flexible cryoprobe has a good diagnostic yield and might be an alternative to consider in cases of diffuse ILD in which a histologic sample is required for diagnosis. This technique could avoid a large number of surgical biopsies.

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): Proposal for a new diagnostic paradigm.

The diagnosis of von Willebrand disease (VWD) remains difficult in a significant proportion of patients. A Spanish multicentre study investigated a cohort of 556 patients from 330 families who were analysed centrally. VWD was confirmed in 480. Next generation sequencing (NGS) of the whole coding VWF was carried out in all recruited patients, compared with the phenotype, and a final diagnosis established. A total of 238 different VWF mutations were found, 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 463 were found to have VWF mutation/s. A good phenotypic/genotypic association was estimated in 96.5% of the patients. One hundred seventy-four patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred sixteen patients presented with mutations that had previously been associated with increased von Willebrand factor (VWF) clearance. RIPA unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 70 patients. The NGS study facilitated an appropriate classification in 63 of them. The remaining seven patients presented with a VWF novel mutation pending further investigation. In five patients with a type 3 and two with a type 2A or 2B phenotype with no mutation, an acquired von Willebrand syndrome (AVWS) was suspected/confirmed. These data seem to support NGS as a first line efficient and faster paradigm in VWD diagnosis.