Dynamic multilayer functional connectivity detects preclinical and clinical Alzheimer's disease.

Increasing evidence suggests that patients with Alzheimer's disease present alterations in functional connectivity but previous results have not always been consistent. One of the reasons that may account for this inconsistency is the lack of consideration of temporal dynamics. To address this limitation, here we studied the dynamic modular organization on resting-state functional magnetic resonance imaging across different stages of Alzheimer's disease using a novel multilayer brain network approach. Participants from preclinical and clinical Alzheimer's disease stages were included. Temporal multilayer networks were used to assess time-varying modular organization. Logistic regression models were employed for disease stage discrimination, and partial least squares analyses examined associations between dynamic measures with cognition and pathology. Temporal multilayer functional measures distinguished all groups, particularly preclinical stages, overcoming the discriminatory power of risk factors such as age, sex, and APOE ϵ4 carriership. Dynamic multilayer functional measures exhibited strong associations with cognition as well as amyloid and tau pathology. Dynamic multilayer functional connectivity shows promise as a functional imaging biomarker for both early- and late-stage Alzheimer's disease diagnosis.

Optical coherence tomography biomarkers in MYO7A-inherited retinal dystrophy: longitudinal study in pediatric patients.

PURPOSE: This study aims to answer a key question: is MYO7A-inherited retinal dystrophy (MYO7A-IRD) a photoreceptor-first or retinal pigment epithelium-first disease? A second aim was to determine the most useful biomarkers to monitor disease progression in pediatric patients with Usher syndrome type 1B (USH1) secondary to MYO7A mutation. METHODS: Fifty-two eyes from 26 patients with genetically-confirmed MYO7A-IRD underwent swept-source optical coherence tomography (SS-OCT). Structural abnormalities were evaluated and correlated with follow-up time and best corrected visual acuity (BCVA). All patients were evaluated at baseline and after ≥ 40 months of follow-up. RESULTS: The mean (SD) patient age was 9.92 (± 4.1) years. Mean follow-up time was 43 (± 3.2) months. At the final evaluation, the most common qualitative abnormalities in the subfoveal area were alterations in the photoreceptor outer segments (76.9% of eyes) and in the interdigitation zone (IZ) (80.8%). The presence of cystoid macular edema at baseline was independently associated with worse BCVA at the final assessment (increase in LogMAR estimate = 0.142; t(45.00) = 2.78, p = 0.009). The mean width of the ellipsoid and interdigitation zones decreased significantly (by 668 µm and 278 µm, respectively; both p < 0.001). CONCLUSION: This study shows that disruption of the photoreceptor outer segments and the IZ are the first alterations detected by SS-OCT in the early phases of MYO7A-IRD. These data highlight the potential value of measuring the width of the ellipsoid and IZ to evaluate disease progression. These findings also demonstrate the utility of monitoring for the emergence of cystic lesions as biomarkers of worse visual prognosis in patients with MYO7A-IRD.

Multiplex Connectome Changes across the Alzheimer's Disease Spectrum Using Gray Matter and Amyloid Data.

The organization of the Alzheimer's disease (AD) connectome has been studied using graph theory using single neuroimaging modalities such as positron emission tomography (PET) or structural magnetic resonance imaging (MRI). Although these modalities measure distinct pathological processes that occur in different stages in AD, there is evidence that they are not independent from each other. Therefore, to capture their interaction, in this study we integrated amyloid PET and gray matter MRI data into a multiplex connectome and assessed the changes across different AD stages. We included 135 cognitively normal (CN) individuals without amyloid-ß pathology (Aß-) in addition to 67 CN, 179 patients with mild cognitive impairment (MCI) and 132 patients with AD dementia who all had Aß pathology (Aß+) from the Alzheimer's Disease Neuroimaging Initiative. We found widespread changes in the overlapping connectivity strength and the overlapping connections across Aß-positive groups. Moreover, there was a reorganization of the multiplex communities in MCI Aß + patients and changes in multiplex brain hubs in both MCI Aß + and AD Aß + groups. These findings offer a new insight into the interplay between amyloid-ß pathology and brain atrophy over the course of AD that moves beyond traditional graph theory analyses based on single brain networks.

Response Variability to Drug Testing in Two Models of Chemically Induced Colitis.

The lack of knowledge regarding the pathogenesis of IBD is a challenge for the development of more effective and safer therapies. Although in vivo preclinical approaches are critical for drug testing, none of the existing models accurately reproduce human IBD. Factors that influence the intra-individual response to drugs have barely been described. With this in mind, our aim was to compare the anti-inflammatory efficacy of a new molecule (MTADV) to that of corticosteroids in TNBS and DSS-induced colitis mice of both sexes in order to clarify further the response mechanism involved and the variability between sexes. The drugs were administered preventively and therapeutically, and real-time bioluminescence was performed for the in vivo time-course colitis monitoring. Morphometric data were also collected, and colonic cytokines and acute plasma phase proteins were analyzed by qRT-PCR and ELISA, respectively-bioluminescence images correlated with inflammatory markers. In the TNBS model, dexamethasone worked better in females, while MTADV improved inflammation in males. In DSS-colitis, both therapies worked similarly. Based on the molecular profiles, interaction networks were constructed to pinpoint the drivers of therapeutic response that were highly dependent on the sex. In conclusion, our results suggest the importance of considering sex in IBD preclinical drug screening.

Palmitoylation of Voltage-Gated Ion Channels.

Protein lipidation is one of the most common forms of posttranslational modification. This alteration couples different lipids, such as fatty acids, phospho- and glycolipids and sterols, to cellular proteins. Lipidation regulates different aspects of the protein's physiology, including structure, stability and affinity for cellular membranes and protein-protein interactions. In this scenario, palmitoylation is the addition of long saturated fatty acid chains to amino acid residues of the proteins. The enzymes responsible for this modification are acyltransferases and thioesterases, which control the protein's behavior by performing a series of acylation and deacylation cycles. These enzymes target a broad repertoire of substrates, including ion channels. Thus, protein palmitoylation exhibits a pleiotropic role by differential modulation of the trafficking, spatial organization and electrophysiological properties of ion channels. Considering voltage-gated ion channels (VGICs), dysregulation of lipidation of both the channels and the associated ancillary subunits correlates with the development of various diseases, such as cancer or mental disorders. Therefore, a major role for protein palmitoylation is currently emerging, affecting not only the dynamism and differential regulation of a moiety of cellular proteins but also linking to human health. Therefore, palmitoylation of VGIC, as well as related enzymes, constitutes a novel pharmacological tool for drug development to target related pathologies.

Pressure injuries during the SARS-CoV-2 pandemic: A retrospective, case-control study.

AIM OF STUDY: The main objective of this study was to ascertain whether severe alterations in hypoxemic, inflammatory, and nutritional parameters in patients diagnosed with SARS-CoV-2 infection were associated with the occurrence and severity of developed dependency-related injuries. The secondary objective was to determine whether there were prognostic factors associated with the occurrence and severity of developed dependency-related injuries during the SARS-CoV-2 pandemic. MATERIAL AND METHODS: A retrospective, single-centre, case-control study was conducted to compare SARS-CoV-2 patients who developed dependency-related injuries after the first 48 h after admission with a control group made up of SARS-CoV-2 patients without dependency-related injuries. The cases of the 1987 patients diagnosed with SARS-CoV-2 infection during the study period were reviewed. Data from 94 patients who developed dependency-related injuries and from 190 patients who did not develop them during hospital admission were analysed. RESULTS: High baseline dependency levels, prolonged hospital stays, and low oxygen saturation levels on arrival in emergency department triage were associated with the occurrence of dependency-related injuries among patients diagnosed with SARS-CoV-2 infection. CONCLUSIONS: SARS-CoV-2 infection can lead to complications such as dependency-related injuries. Although there are several non-modifiable variables associated with the occurrence of dependency-related injuries in these patients, it is essential to conduct further research and introduce consensus guidelines to reduce their incidence and prevalence.

[Pneumatosis cystoides intestinalis complicated with intestinal volvulus]. / Neumatosis quística intestinal complicada con vólvulo intestinal.

Pneumatosis cystoides intestinalis is a rare condition in which multiple gas-filled cysts are found within the wall of the gastrointestinal tract either in the subserosa or submucosa. Its pathogenesis is uncertain and several pathogenic mechanisms have been proposed to explain its origin. The case of a male patient of 46 years with previous diagnosis of pneumatosis cystic intestinalis, who consulted for abdominal pain, vomiting and fever (39 °C) is presented. By the time of admission ther were signs of peritoneal irritation. The X-ray abdominal reported distension and intestinal hydro-air levels. Exploratory laparotomy was performed and revealed small bowel volvulus with strangulation of some intestinal segment. Histological diagnosis was pneumatosis cystic intestinalis complicated with Infarction trans-mural by intestinal volvulus. The patient evolved satisfactorily.

Jejunal administration of glucose enhances acyl ghrelin suppression in obese humans.

Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (iv) infusions of glucose. These "isoglycemic clamps" enabled us to determine the contribution of the GI tract and postabsorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared with gastric glucose administration (P < 0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P ≤ 0.01). However, gastric and isoglycemic iv glucose infusions resulted in similar degrees of acyl ghrelin suppression (P > 0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion.

Linking structural and functional changes during aging using multilayer brain network analysis.

Brain structure and function are intimately linked, however this association remains poorly understood and the complexity of this relationship has remained understudied. Healthy aging is characterised by heterogenous levels of structural integrity changes that influence functional network dynamics. Here, we use the multilayer brain network analysis on structural (diffusion weighted imaging) and functional (magnetoencephalography) data from the Cam-CAN database. We found that the level of similarity of connectivity patterns between brain structure and function in the parietal and temporal regions (alpha frequency band) is associated with cognitive performance in healthy older individuals. These results highlight the impact of structural connectivity changes on the reorganisation of functional connectivity associated with the preservation of cognitive function, and provide a mechanistic understanding of the concepts of brain maintenance and compensation with aging. Investigation of the link between structure and function could thus represent a new marker of individual variability, and of pathological changes.

Kv1.3 in the spotlight for treating immune diseases.

INTRODUCTION: Kv1.3 is the main voltage-gated potassium channel of leukocytes from both the innate and adaptive immune systems. Channel function is required for common processes such as Ca2+ signaling but also for cell-specific events. In this context, alterations in Kv1.3 are associated with multiple immune disorders. Excessive channel activity correlates with numerous autoimmune diseases, while reduced currents result in increased cancer prevalence and immunodeficiencies. AREAS COVERED: This review offers a general view of the role of Kv1.3 in every type of leukocyte. Moreover, diseases stemming from dysregulations of the channel are detailed, as well as current advances in their therapeutic research. EXPERT OPINION: Kv1.3 arises as a potential immune target in a variety of diseases. Several lines of research focused on channel modulation have yielded positive results. However, among the great variety of specific channel blockers, only one has reached clinical trials. Future investigations should focus on developing simpler administration routes for channel inhibitors to facilitate their entrance into clinical trials. Prospective Kv1.3-based treatments will ensure powerful therapies while minimizing undesired side effects.

Synthesis of Cobalt-Based Nanoparticles as Catalysts for Methanol Synthesis from CO2 Hydrogenation.

The increasing emission of carbon dioxide into the atmosphere has urged the scientific community to investigate alternatives to alleviate such emissions, being that they are the principal contributor to the greenhouse gas effect. One major alternative is carbon capture and utilization (CCU) toward the production of value-added chemicals using diverse technologies. This work aims at the study of the catalytic potential of different cobalt-derived nanoparticles for methanol synthesis from carbon dioxide hydrogenation. Thanks to its abundance and cost efficacy, cobalt can serve as an economical catalyst compared to noble metal-based catalysts. In this work, we present a systematic comparison among different cobalt and cobalt oxide nanocomposites in terms of their efficiency as catalysts for carbon dioxide hydrogenation to methanol as well as how different supports, zeolites, MnO2, and CeO2, can enhance their catalytic capacity. The oxygen vacancies in the cerium oxide act as carbon dioxide adsorption and activation sites, which facilitates a higher methanol production yield.

Deciphering complexity: TULP1 variants linked to an atypical retinal dystrophy phenotype.

Introduction: TULP1 exemplifies the remarkable clinical and genetic heterogeneity observed in inherited retinal dystrophies. Our research describes the clinical and molecular characteristics of a patient manifesting an atypical retinal dystrophy pattern, marked by the identification of both a previously unreported and a rarely encountered TULP1 variant. Methods: Whole-exome sequencing was performed to identify potential causative variants. The pathogenicity of the identified TULP1 variants was evaluated through in silico predictors and a minigene splice assay, specifically designed to assess the effect of the unreported TULP1 variant. Results: We identified two TULP1 gene variants in a patient exhibiting unusual and symmetrical alterations in both retinas, characterized by an increase in autofluorescence along the distribution of retinal vessels. These variants included a known rare missense variant, c.1376T>C, and a novel splice site variant, c.822G>T. For the latter variant (c.822G>T), we conducted a minigene splice assay that demonstrated the incorporation of a premature stop codon. This finding suggests a likely activation of the nonsense-mediated mRNA decay mechanism, ultimately resulting in the absence of protein production from this allele. Segregation analysis confirmed that these variants were in trans. Discussion: Our data support that individuals with biallelic TULP1 variants may present with a unique pattern of macular degeneration and periarteriolar vascular pigmentation. This study highlights the importance of further clinical and molecular characterization of TULP1 variants to elucidate genotype-phenotype correlations in the context of inherited retinal dystrophies.

Optical Coherence Tomography in Inherited Macular Dystrophies: A Review.

Macular dystrophies (MDs) constitute a collection of hereditary retina disorders leading to notable visual impairment, primarily due to progressive macular atrophy. These conditions are distinguished by bilateral and relatively symmetrical abnormalities in the macula that significantly impair central visual function. Recent strides in fundus imaging, especially optical coherence tomography (OCT), have enhanced our comprehension and diagnostic capabilities for MD. OCT enables the identification of neurosensory retinal disorganization patterns and the extent of damage to retinal pigment epithelium (RPE) and photoreceptor cells in the dystrophies before visible macular pathology appears on fundus examinations. It not only helps us in diagnostic retinal and choroidal pathologies but also guides us in monitoring the progression of, staging of, and response to treatment. In this review, we summarize the key findings on OCT in some of the most common MD.

Vagus nerve dysfunction in the post-COVID-19 condition: a pilot cross-sectional study.

OBJECTIVES: The post-COVID-19 condition (PCC) is a disabling syndrome affecting at least 5%-10% of subjects who survive COVID-19. SARS-CoV-2 mediated vagus nerve dysfunction could explain some PCC symptoms, such as dysphonia, dysphagia, dyspnea, dizziness, tachycardia, orthostatic hypotension, gastrointestinal disturbances, or neurocognitive complaints. METHODS: We performed a cross-sectional pilot study in subjects with PCC with symptoms suggesting vagus nerve dysfunction (n = 30) and compared them with subjects fully recovered from acute COVID-19 (n = 14) and with individuals never infected (n = 16). We evaluated the structure and function of the vagus nerve and respiratory muscles. RESULTS: Participants were mostly women (24 of 30, 80%), and the median age was 44 years (interquartile range [IQR] 35-51 years). Their most prevalent symptoms were cognitive dysfunction 25 of 30 (83%), dyspnea 24 of 30 (80%), and tachycardia 24 of 30 (80%). Compared with COVID-19-recovered and uninfected controls, respectively, subjects with PCC were more likely to show thickening and hyperechogenic vagus nerve in neck ultrasounds (cross-sectional area [CSA] [mean ± standard deviation]: 2.4 ± 0.97mm2 vs. 2 ± 0.52mm2 vs. 1.9 ± 0.73 mm2; p 0.08), reduced esophageal-gastric-intestinal peristalsis (34% vs. 0% vs. 21%; p 0.02), gastroesophageal reflux (34% vs. 19% vs. 7%; p 0.13), and hiatal hernia (25% vs. 0% vs. 7%; p 0.05). Subjects with PCC showed flattening hemidiaphragms (47% vs. 6% vs. 14%; p 0.007), and reductions in maximum inspiratory pressure (62% vs. 6% vs. 17%; p ≤ 0.001), indicating respiratory muscle weakness. The latter findings suggest additional involvement of the phrenic nerve. DISCUSSION: Vagus and phrenic nerve dysfunction contribute to the complex and multifactorial pathophysiology of PCC.

Directed Functional Brain Connectivity is Altered in Sub-threshold Amyloid-ß Accumulation in Cognitively Normal Individuals.

Several studies have shown that amyloid-ß (Aß) deposition below the clinically relevant cut-off levels is associated with subtle changes in cognitive function and increases the risk of developing future Alzheimer's disease (AD). Although functional MRI is sensitive to early alterations occurring during AD, sub-threshold changes in Aß levels have not been linked to functional connectivity measures. This study aimed to apply directed functional connectivity to identify early changes in network function in cognitively unimpaired participants who, at baseline, exhibit Aß accumulation below the clinically relevant threshold. To this end, we analyzed baseline functional MRI data from 113 cognitively unimpaired participants of the Alzheimer's Disease Neuroimaging Initiative cohort who underwent at least one 18F-florbetapir-PET after the baseline scan. Using the longitudinal PET data, we classified these participants as Aß negative (Aß-) non-accumulators (n = 46) and Aß- accumulators (n = 31). We also included 36 individuals who were amyloid-positive (Aß+) at baseline and continued to accumulate Aß (Aß+ accumulators). For each participant, we calculated whole-brain directed functional connectivity networks using our own anti-symmetric correlation method and evaluated their global and nodal properties using measures of network segregation (clustering coefficient) and integration (global efficiency). When compared to Aß- non-accumulators, the Aß- accumulators showed lower global clustering coefficient. Moreover, the Aß+ accumulator group exhibited reduced global efficiency and clustering coefficient, which at the nodal level mainly affected the superior frontal gyrus, anterior cingulate cortex, and caudate nucleus. In Aß- accumulators, global measures were associated with lower baseline regional PET uptake values, as well as higher scores on the Modified Preclinical Alzheimer Cognitive Composite. Our findings indicate that directed connectivity network properties are sensitive to subtle changes occurring in individuals who have not yet reached the threshold for Aß positivity, which makes them a potentially viable marker to detect negative downstream effects of very early Aß pathology.

Nonlinear changes in delayed functional network topology in Alzheimer's disease: relationship with amyloid and tau pathology.

BACKGROUND: Alzheimer's disease is a neurodegenerative disorder associated with the abnormal deposition of pathological processes, such as amyloid-ß and tau, which produces nonlinear changes in the functional connectivity patterns between different brain regions across the Alzheimer's disease continuum. However, the mechanisms underlying these nonlinear changes remain largely unknown. Here, we address this question using a novel method based on temporal or delayed correlations and calculate new whole-brain functional networks to tackle these mechanisms. METHODS: To assess our method, we evaluated 166 individuals from the ADNI database, including amyloid-beta negative and positive cognitively normal subjects, patients with mild cognitive impairment, and patients with Alzheimer's disease dementia. We used the clustering coefficient and the global efficiency to measure the functional network topology and assessed their relationship with amyloid and tau pathology measured by positron emission tomography, as well as cognitive performance using tests measuring memory, executive function, attention, and global cognition. RESULTS: Our study found nonlinear changes in the global efficiency, but not in the clustering coefficient, showing that the nonlinear changes in functional connectivity are due to an altered ability of brain regions to communicate with each other through direct paths. These changes in global efficiency were most prominent in early disease stages. However, later stages of Alzheimer's disease were associated with widespread network disruptions characterized by changes in both network measures. The temporal delays required for the detection of these changes varied across the Alzheimer's disease continuum, with shorter delays necessary to detect changes in early stages and longer delays necessary to detect changes in late stages. Both global efficiency and clustering coefficient showed quadratic associations with pathological amyloid and tau burden as well as cognitive decline. CONCLUSIONS: This study suggests that global efficiency is a more sensitive indicator of network changes in Alzheimer's disease when compared to clustering coefficient. Both network properties were associated with pathology and cognitive performance, demonstrating their relevance in clinical settings. Our findings provide an insight into the mechanisms underlying nonlinear changes in functional network organization in Alzheimer's disease, suggesting that it is the lack of direct connections that drives these functional changes.

Sex differences in multilayer functional network topology over the course of aging in 37543 UK Biobank participants.

Aging is a major risk factor for cardiovascular and neurodegenerative disorders, with considerable societal and economic implications. Healthy aging is accompanied by changes in functional connectivity between and within resting-state functional networks, which have been associated with cognitive decline. However, there is no consensus on the impact of sex on these age-related functional trajectories. Here, we show that multilayer measures provide crucial information on the interaction between sex and age on network topology, allowing for better assessment of cognitive, structural, and cardiovascular risk factors that have been shown to differ between men and women, as well as providing additional insights into the genetic influences on changes in functional connectivity that occur during aging. In a large cross-sectional sample of 37,543 individuals from the UK Biobank cohort, we demonstrate that such multilayer measures that capture the relationship between positive and negative connections are more sensitive to sex-related changes in the whole-brain connectivity patterns and their topological architecture throughout aging, when compared to standard connectivity and topological measures. Our findings indicate that multilayer measures contain previously unknown information on the relationship between sex and age, which opens up new avenues for research into functional brain connectivity in aging.

The Phosphorylation of Kv1.3: A Modulatory Mechanism for a Multifunctional Ion Channel.

The voltage-gated potassium channel Kv1.3 plays a pivotal role in a myriad of biological processes, including cell proliferation, differentiation, and apoptosis. Kv1.3 undergoes fine-tuned regulation, and its altered expression or function correlates with tumorigenesis and cancer progression. Moreover, posttranslational modifications (PTMs), such as phosphorylation, have evolved as rapid switch-like moieties that tightly modulate channel activity. In addition, kinases are promising targets in anticancer therapies. The diverse serine/threonine and tyrosine kinases function on Kv1.3 and the effects of its phosphorylation vary depending on multiple factors. For instance, Kv1.3 regulatory subunits (KCNE4 and Kvß) can be phosphorylated, increasing the complexity of channel modulation. Scaffold proteins allow the Kv1.3 channelosome and kinase to form protein complexes, thereby favoring the attachment of phosphate groups. This review compiles the network triggers and signaling pathways that culminate in Kv1.3 phosphorylation. Alterations to Kv1.3 expression and its phosphorylation are detailed, emphasizing the importance of this channel as an anticancer target. Overall, further research on Kv1.3 kinase-dependent effects should be addressed to develop effective antineoplastic drugs while minimizing side effects. This promising field encourages basic cancer research while inspiring new therapy development.

Improving Hereditary Hemorrhagic Telangiectasia Molecular Diagnosis: A Referral Center Experience.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disease inherited in an autosomal dominant manner. Disease-causing variants in endoglin (ENG) and activin A receptor type II-like 1 (ACVRL1) genes are detected in more than 90% of the patients undergoing molecular testing. The identification of variants of unknown significance is often seen as a challenge in clinical practice that makes family screening and genetic counseling difficult. Here, we show that the implementation of cDNA analysis to assess the effect of splice site variants on mRNA splicing is a powerful tool. METHODS: Gene panel sequencing of genes associated with HHT and other arteriovenous malformation-related syndromes was performed. To evaluate the effect of the splice site variants, cDNA analysis of ENG and ACVRL1 genes was carried out. RESULTS: three novel splice site variants were identified in ENG (c.68-2A > T and c.1311+4_1311+8del) and ACVLR1 (c.526-6C > G) genes correspondingly in three individuals with HHT that met ≥ 3 Curaçao criteria. All three variants led to an aberrant splicing inducing exon skipping (ENG:c.68-2A > T and ACVRL1:c.526-6C > G) or intron retention (ENG:c.1311+4_1311+8del) allowing the confirmation of the predicted effect on splicing and the reclassification from unknown significance to pathogenic/likely pathogenic of two of them. CONCLUSIONS: RNA analysis should be performed to assess and/or confirm the impact of variants on splicing. The molecular diagnosis of HHT patients is crucial to allow family screening and accurate genetic counseling. A multidisciplinary approach including clinicians and geneticists is crucial when dealing with patients with rare diseases.